Circovirus sequences associated with piglet weight loss disease (pwd)

ABSTRACT

The genome sequences and the nucleotide sequences coding for the PWD circovirus polypeptides, such as the circovirus structural and non-structural polypeptides, vectors including the sequences, and cells and animals transformed by the vectors are provided. Methods for detecting the nucleic acids or polypeptides, and kits for diagnosing infection by a PWD circovirus, also are provided. Method for selecting compounds capable of modulating the viral infection are further provided. Pharmaceutical, including vaccine, compositions for preventing and/or treating viral infections caused by PWD circovirus and the use of vectors for preventing and/or treating diseases also are provided.

INFORMATION ON RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/588,237, filed Oct. 27, 2006, now U.S. Pat. No. ______, which is adivisional of U.S. patent application Ser. No. 10/718,264, filed Nov.21, 2003, now U.S. Pat. No. 7,179,472, which is a divisional of U.S.patent application Ser. No. 09/514,245, filed Feb. 28, 2000, now U.S.Pat. No. 6,703,023, which is a continuation-in-part of InternationalPatent Application No. PCT/FR98/02634, filed Dec. 4, 1998, published ina non-English language, and now abandoned, which claims priority toFrench Application No. 97/15396, filed Dec. 5, 1997, the specificationsof which are incorporated herein by reference in their entireties forall purposes.

BACKGROUND OF THE INVENTION

The invention relates to the genomic sequence and nucleotide sequencescoding for polypeptides of PWD circovirus, such as the structural andnonstructural polypeptides of said circovirus, as well as vectorsincluding said sequences and cells or animals transformed by thesevectors. The invention likewise relates to methods for detecting thesenucleic acids or polypeptides and kits for diagnosing infection by thePWD circovirus. The invention is also directed to a method for selectingcompounds capable of modulating the viral infection. The inventionfurther comprises pharmaceutical compositions, including vaccines, forthe prevention and/or the treatment of viral infections by PWDcircovirus as well as the use of a vector according to the invention forthe prevention and/or the treatment of diseases by gene therapy.

Piglet weight loss disease (PWD), alternatively called fatal pigletwasting (FPW) has been widely described in North America (Harding, J.C., 1997), and authors have reported the existence of a relationshipbetween this pathology and the presence of porcine circovirus (Daft, B.et al., 1996; Clark, E. G., 1997; Harding, J. C., 1997; Harding, J. C.and Clark, E. G., 1997; Nayar, G. P. et al., 1997). A porcine circovirushas already been demonstrated in established lines of cell culturesderived from pigs and chronically infected (Tischer, I., 1986, 1988,1995; Dulac, G. C., 1989; Edwards, S., 1994; Allan, G. M., 1995 andMcNeilly, F., 1996). This virus, during experimental infection ofpiglets, does not prove pathogenic for pigs (Tischer, I., 1986, Homer,G. W., 1991) and its nucleotide sequence has been determined andcharacterized (Tischer, I., 1982; Meehan, B. M. et al., 1997; Mankertz.,A., 1997). The porcine circovirus, called PCV virus, is part of thecircovirus genus of the circoviridae family (Murphy, F. A. et al., 1995)whose virion has a circular DNA of size between 1.7 and 2.3 kb, whichDNA comprises three open reading frames (ORF1 to ORF3), coding for areplication protein REP involved in the initiation and termination phaseof rolling circular replication (RCR) (Heyraud-Nitschke, F., et al.,1995; Harding, M. R. et al., 1993; Hanson, S. F. et al., 1995; Fontes,E. P. B. et al., 1994), coding for a capsid protein (Boulton, L. H. etal., 1997; Hackland, A. F. et al., 1994; Chu, P. W. G. et al., 1993) andcoding for a nonstructural protein called a dissemination protein(Lazarowitz., S. G. et al., 1989).

The inventors of the present invention have noticed that the clinicalsigns perceptible in pigs and linked to infection by the PWD circovirusare very distinctive. These manifestations in general appear in pigs of8 to 12 weeks of age, weaned for 4 to 8 weeks. The first signs arehypotonia without it being possible to speak of prostration. Rapidly (48hours), the flanks hollow, the line of the spine becomes apparent, andthe pigs “blanch.” These signs are in general accompanied byhyperthermia, anorexia and most often by respiratory signs (coughing,dyspnea, polypnea). Transitory diarrhea can likewise appear. The diseasestate phase lasts approximately one month at the end of which the rateof mortality varies from 5 to 20%. To these mortalities, it is expedientto add a variable proportion (5-10%) of cadaveric animals which are nolonger able to present an economic future. It is to be noted thatoutside of this critical stage of the end of post-weaning, no anomalyappears on the farms. In particular, the reproductive function istotally maintained.

On the epidemiological level, the first signs of this pathology appearedat the start of 1995 in the east of the Côtes d'Armor region in France,and the farms affected are especially confined to this area of theregion. In December 1996, the number of farms concerned could not beevaluated with precision because of the absence of a specific laboratorydiagnostic method or of an epidemiological surveillance system of thelivestock. Based on the clinical facts as well as on results ofpostmortem examinations supplied by veterinarians, it is possible toestimate this number as several dozen (80-100). The contagiousness ofthe disease is weak to moderate. Cases are being reported outside theinitial area and for the majority are following the transfer of animalscoming from farms familiar with the problem. On the other hand, acharacteristic of the condition is its strong remanence. Thus, farmswhich have been affected for a year are still affected in spite of themassive administration of therapeutics. Farms with clinical expressionare drawn from various categories of specialization (breeders/fatteners,post-weaners/ fatteners) and different economic structures areconcerned. In addition, the disorders appear even in farms where therules of animal husbandry are respected.

Numerous postmortem examinations have been carried out either on farmsor in the laboratory. The elements of the lesional table are disparate.The most constant macroscopic lesions are pneumonia which sometimesappears in patchy form as well as hypertrophy of the lymphatic ganglia.The other lesions above all affect the thoracic viscera including,especially, pericarditis and pleurisy. However, arthritis and gastriculcers are also observed. The lesions revealed in the histologicalexamination are essentially situated at the pulmonary level(interstitial pneumonia), ganglionic level (lymphoid depletion of thelymph nodes, giant cells) and renal level (glomerulonephritis,vasculitis). The infectious agents have been the subject of wideresearch. It has been possible to exclude the intervention ofpestiviruses and Aujeszky's disease. The disorders appear in theseropositive PDRS (Porcine Dysgenic and Respiratory Syndrome, aninfection linked to an arteriovirus) herds, but it has not been possibleto establish the role of the latter in the genesis of the disorders (themajority of the farms in Brittany are PDRS seropositive).

The inventors of the present invention, with the aim of identifying theetiological agent responsible for PWD, have carried out “contact” testsbetween piglets which are obviously “ill” and SPF pigs (specificpathogen-free) from CNEVA (Centre National d′Etudes Vétérinaires etAlimentaires, France). These tests allow the development of signscomparable to those observed on the farm to be observed in protectedanimal houses. The discrete signs such as moderate hyperthermia,anorexia and intermittent diarrhea appeared after one week of contact.It must be noted that the PDRS virus only diffused subsequent to theclinical signs. In addition, inoculations of organ homogenates of sickanimals to healthy pigs allowed signs related to those observed on thefarms to be reproduced, although with a lower incidence, linked to thefavorable conditions of upkeep of the animals in the experimentalinstallations.

Thus, the inventors of the present invention have been able todemonstrate that the pathological signs appear as a well-defined entityaffecting the pig at a particular stage of its growth.

This pathology has never been described in France. However, sparseinformation, especially Canadian, relates to similar facts.

The disorders cannot be mastered with the existing therapeutics.

The data collected both on the farm and by experimentation have allowedthe following points to be highlighted:

-   -   PWD is transmissible but its contagiousness is not very high,    -   its etiological origin is of infectious and probably viral        nature,    -   PWD has a persistent character in the affected farms.

Considerable economic consequences ensue for the farms.

Thus, there is currently a significant need for a specific and sensitivediagnostic, whose production is practical and rapid, allowing the earlydetection of the infection.

A reliable, sensitive and practical test which allows the distinctionbetween strains of porcine circovirus (PCV) is thus strongly desirable.

On the other hand, a need for efficient and well-tolerated treatment ofinfections with PWD circovirus likewise remains desirable, no vaccinecurrently being available against PWD circovirus.

Concerning PWD circovirus, it will probably be necessary to understandthe role of the immune defense in the physiology and the pathology ofthe disease to develop satisfactory vaccines.

Fuller information concerning the biology of these strains, theirinteractions with their hosts, the associated infectivity phenomena andthose of escape from the immune defenses of the host especially, andfinally their implication in the development of associated pathologies,will allow a better understanding of these mechanisms. Taking intoaccount the facts which have been mentioned above and which show inparticular the limitations of combating infection by the PWD circovirus,it is thus essential today on the one hand to develop molecular tools,especially starting from a better genetic knowledge of the PWDcircovirus, and likewise to perfect novel preventive and therapeutictreatments, novel methods of diagnosis and specific, efficacious andtolerated novel vaccine strategies. This is precisely the subject of thepresent invention.

SUMMARY OF THE INVENTION

The present invention relates to vaccines comprising a nucleotidesequence of the genome of Porcine circovirus type B, or a homologue orfragment thereof, and an acceptable pharmaceutical or veterinaryvehicle. In one embodiment of the invention, the nucleotide sequence isselected from SEQ ID No. 15, SEQ ID No. 19 SEQ ID No. 23, or SEQ ID No.25, or a homologue or fragment thereof. In another embodiment of theinvention, the homologue has at least 80% sequence identity to SEQ IDNo. 15, SEQ ID No. 19, SEQ ID No. 23 or SEQ ID No. 25. In yet anotherembodiment, the vaccines further comprising an adjuvant

The present invention also relates to vaccines comprising a polypeptideencoded by a nucleotide sequence of the genome of PCVB, or a homologueor fragment thereof, and an acceptable pharmaceutical or veterinaryvehicle. In one embodiment, the homologue has at least 80% sequenceidentity to SEQ ID No. 15, SEQ ID No. 19, SEQ ID No. 23 or SEQ ID No.25. In another embodiment of the invention, the nucleotide sequence isselected from SEQ ID No. 23 or SEQ ID No. 25, or a homologue or fragmentthereof. In still another embodiment, the polypeptide has the amino acidsequence of SEQ ID No. 24 or SEQ ID No. 26. In yet another embodiment,the homologue has at least 80% sequence identity to SEQ ID No. 24 or SEQID No. 26. In another embodiment, the polypeptide has the amino acidsequence of SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, or SEQ ID No.32.

A further aspect of the invention relates to vaccines comprising avector and an acceptable pharmaceutical or veterinary vehicle, thevector comprising a nucleotide sequence of the genome of Porcinecircovirus type B, or a homologue or fragment thereof. In oneembodiment, the vaccine further comprises a gene coding for anexpression product capable of inhibiting or retarding the establishmentor development of a genetic or acquired disease.

The present invention also relates to vaccines comprising a cell and anacceptable pharmaceutical or veterinary vehicle, wherein the cell istransformed with a nucleotide sequence of the genome of Porcinecircovirus type B, or a homologue or fragment thereof.

Still further, the present invention relates to vaccines comprising apharmaceutically acceptable vehicle and a single polypeptide, whereinthe single polypeptide consists of SEQ ID No. 26.

Additionally, the present invention relates to methods of immunizing amammal against piglet weight loss disease comprising administering to amammal an effective amount of the vaccines described above.

These and other aspects of the invention will become apparent to theskilled artisan in view of the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Experimental scheme which has made it possible to bring aboutthe isolation and the identification of the circovirus associated withPWD of type A and B.

Test 1: experimental reproduction of the PWD by inoculation of pig organhomogenates from farms affected by PWD.

Test 2: experimental reproduction of PWD.

Test 3: experimental reproduction of PWD.

Test 4: no experimental reproduction of PWD.

FIG. 2: Organization of the genome of the circovirus associated with PWDof type A (PCVA)

strand of (+) polarity (SEQ ID No. 1);

strand of (−) polarity (SEQ ID No. 5, represented according to theorientation 3′→5′);

sequences of amino acids of proteins encoded by the two DNA strands inthe three possible reading frames SEQ ID NOS: 2-4 and 6-8 respectively.

FIG. 3: Alignment of the nucleotide sequence SEQ ID No. 1 of the PWDcircovirus of type A (PCVA) and of the MEEHAN SEQ ID No. 163 strain andMANKERTZ SEQ ID No. 164 strain circoviruses of the porcine cell lines.

FIG. 4: Alignment of the sequence of amino acids SEQ ID No. 10 of apolypeptide encoded by the nucleotide sequence SEQ ID No. 9 (ORF1) ofthe PWD circovirus of type A (PCVA) and of corresponding nucleotidesequences of the MEEHAN SEQ ID No. 165 strain and MANKERTZ SEQ ID No.166 strain circoviruses of the porcine cell lines.

FIG. 5: Alignment of the sequence of amino acids SEQ ID No. 12 of apolypeptide encoded by the nucleotide sequence SEQ ID No. 11 (ORF2) ofthe PWD circovirus of type A (PCVA) and of corresponding nucleotidesequences of the MEEHAN SEQ ID No. 167 strain and MANKERTZ SEQ ID No.168 strain circoviruses of the porcine cell lines.

FIG. 6: Alignment of the sequence of amino acids SEQ ID No. 14 of apolypeptide encoded by the nucleotide sequence SEQ ID No. 13 (ORF3) ofthe PWD circovirus of type A (PCVA) and of corresponding nucleotidesequences of the MEEHAN SEQ ID No. 169 strain and MANKERTZ SEQ ID No.170 strain circoviruses of the porcine cell lines.

FIG. 7: Western blot analysis of recombinant proteins of the PWDcircovirus of type A (PCVA).

The analyses were carried out on cell extracts of Sf9 cells obtainedafter infection with recombinant baculovirus PCF ORF 1.

FIG. 8: Organization of the genome of the circovirus associated with thePWD of type B (PCVB)

strand of (+) polarity (SEQ ID No. 15);

strand of (−) polarity (SEQ ID No. 19, represented according to theorientation 3′→5′);

sequence of amino acids of proteins encoded by the two DNA strands inthe three possible reading frames SEQ ID NOS: 16-18 and 20-22respectively.

FIG. 9: Evolution of the daily mean gain (DMG) of pig farms affected bypiglet weight loss disease (PWD), placed under experimental conditions.

FIG. 10: DMG compared for the 3 batches of pigs (F1, F3 and F4)calculated over a period of 28 days, after vaccination test.

FIG. 11: Hyperthermia greater than 41° C., expressed as a percentagecompared for the 3 batches of pigs (F1, F3 and F4) calculated per weekover a period of 28 days, after vaccination test.

FIG. 12: Membranes of peptide spots corresponding to the ORF2s revealedwith the aid of an infected pig serum, originating from a conventionalfarm.

The numbers of specific peptides of the circovirus of type B as well astheir nonreactive homologs (type A) are indicated in bold.

The nonspecific immunogenic peptides are indicated in italics.

FIG. 13: Alignment of amino acid sequences of proteins encoded by theORF2 of the PWD circovirus of type A SEQ ID No. 12 and by the ORF′2 ofthe PWD circovirus of type B SEQ ID No. 26. The position of 4 peptidescorresponding to specific epitopes of the PWD circovirus of type B isindicated on the corresponding sequence by a bold line, their homolog onthe sequence of the PWD circovirus of type A is likewise indicated by anordinary line.

FIG. 14: Charts the results of experiments that demonstrate, in terms ofpercent hyperthermia, that vaccination with ORF′1 and ORF′2 of PCV-Benhances the level of protection in swine challenged with PCV-B (Percenthyperthermia: >40.5 C, control: not vaccinated and not challenged,ORF′1: vaccinated and challenged, ORF′2: vaccinated and challenged, ORF:not vaccinated, challenged).

FIG. 15: Charts the results of experiments that demonstrate, in terms ofanimal growth, that vaccination with ORF′1 and ORF′2 of PCV-B enhancesthe level of protection in swine challenged with PCV-B (Control: notvaccinated, not challenged, ORF′1: vaccinated and challenged, ORF′2:vaccinated and challenged, ORF: not vaccinated, challenged).

FIG. 16: Immunoperoxidase staining of PK15 cells at 24 hpost-transfection with the pcDNA3/ORF′2 plasmid. Expression of PCVBORF′2 was confirmed by IPMA following incubation in the presence of theswine anti-PCVB monospecific serum.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nucleotide sequences of the genome ofPWD circovirus selected from the sequences SEQ ID No. 1, SEQ ID No. 5,SEQ ID No. 15, SEQ ID No. 19 or one of their fragments.

The nucleotide sequences of sequences SEQ ID No. 1 and SEQ ID No. 5correspond respectively to the genome sequence of the strand of (+)polarity and of the strand of (−) polarity of the PWD circovirus of typeA (or PCVA), the sequence SEQ ID No. 5 being represented according tothe orientation 5′→3′.

The nucleotide sequences of sequences SEQ ID No. 15 and SEQ ID No. 19correspond respectively to the genome sequence of the strand of (+)polarity and of the strand of (−) polarity of the PWD circovirus of typeB (or PCVB), the sequence SEQ ID No. 19 being represented according tothe orientation 5′→3′.

The present invention likewise relates to nucleotide sequences,characterized in that they are selected from:

-   -   a) a nucleotide sequence of a specific fragment of the sequence        SEQ ID No. 1, SEQ ID No. 5, SEQ ID No. 15, SEQ ID No. 19 or one        of their fragments;    -   b) a nucleotide sequence homologous to a nucleotide sequence        such as defined in a);    -   c) a nucleotide sequence complementary to a nucleotide sequence        such as defined in a) or b), and a nucleotide sequence of their        corresponding RNA;    -   d) a nucleotide sequence capable of hybridizing under stringent        conditions with a sequence such as defined in a), b) or c);    -   e) a nucleotide sequence comprising a sequence such as defined        in a), b), c) or d); and    -   f) a nucleotide sequence modified by a nucleotide sequence such        as defined in a), b), c), d) or e).

Nucleotide, polynucleotide or nucleic acid sequence will be understoodaccording to the present invention as meaning both a double-stranded orsingle-stranded DNA in the monomeric and dimeric (so-called in tandem)forms and the transcription products of said DNAs.

It must be understood that the present invention does not relate to thegenomic nucleotide sequences taken in their natural environment, that isto say in the natural state. It concerns sequences which it has beenpossible to isolate, purify or partially purify, starting fromseparation methods such as, for example, ion-exchange chromatography, byexclusion based on molecular size, or by affinity, or alternativelyfractionation techniques based on solubility in different solvents, orstarting from methods of genetic engineering such as amplification,cloning and subcloning, it being possible for the sequences of theinvention to be carried by vectors.

The nucleotide sequences SEQ ID No. 1 and SEQ ID No. 15 were obtained bysequencing of the genome by the Sanger method.

Nucleotide sequence fragment according to the invention will beunderstood as designating any nucleotide fragment of the PWD circovirus,type A or B, of length of at least 8 nucleotides, preferably at least 12nucleotides, and even more preferentially at least 20 consecutivenucleotides of the sequence from which it originates.

Specific fragment of a nucleotide sequence according to the inventionwill be understood as designating any nucleotide fragment of the PWDcircovirus, type A or B, having, after alignment and comparison with thecorresponding fragments of known porcine circoviruses, at least onenucleotide or base of different nature. For example, the specificnucleotide fragments of the PWD circovirus of type A can easily bedetermined by referring to FIG. 3 of the present invention in which thenucleotides or bases of the sequence SEQ ID No. 1 (circopordfp) areshown which are of different nature, after alignment of said sequenceSEQ ID No. 1 with the other two sequences of known porcine circovirus(circopormeeh and circopormank).

Homologous nucleotide sequence in the sense of the present invention isunderstood as meaning a nucleotide sequence having at least a percentageidentity with the bases of a nucleotide sequence according to theinvention of at least 80%, preferably 90% or 95%, this percentage beingpurely statistical and it being possible to distribute the differencesbetween the two nucleotide sequences at random and over the whole oftheir length.

Specific homologous nucleotide sequence in the sense of the presentinvention is understood as meaning a homologous nucleotide sequencehaving at least one nucleotide sequence of a specific fragment, such asdefined above. Said “specific” homologous sequences can comprise, forexample, the sequences corresponding to the genomic sequence or to thesequences of its fragments representative of variants of PWD circovirusof type A or B. These specific homologous sequences can thus correspondto variations linked to mutations within strains of PWD circovirus oftype A and B, and especially correspond to truncations, substitutions,deletions and/or additions of at least one nucleotide. Said homologoussequences can likewise correspond to variations linked to the degeneracyof the genetic code.

The term “degree or percentage of sequence homology” refers to “degreeor percentage of sequence identity between two sequences after optimalalignment” as defined in the present application.

Two amino-acids or nucleotidic sequences are said to be “identical” ifthe sequence of amino-acids or nucleotidic residues, in the twosequences is the same when aligned for maximum correspondence asdescribed below. Sequence comparisons between two (or more) peptides orpolynucleotides are typically performed by comparing sequences of twooptimally aligned sequences over a segment or “comparison window” toidentify and compare local regions of sequence similarity. Optimalalignment of sequences for comparison may be conducted by the localhomology algorithm of Smith and Waterman, Ad. App. Math 2: 482 (1981),by the homology alignment algorithm of Neddleman and Wunsch, J. Mol.Biol. 48: 443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerizedimplementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group (GCG),575 Science Dr., Madison, Wis.), or by visual inspection.

“Percentage of sequence identity” (or degree or identity) is determinedby comparing two optimally aligned sequences over a comparison window,where the portion of the peptide or polynucleotide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalamino-acid residue or nucleic acid base occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity.

The definition of sequence identity given above is the definition thatwould use one of skill in the art. The definition by itself does notneed the help of any algorithm, said algorithms being helpful only toachieve the optimal alignments of sequences, rather than the calculationof sequence identity.

From the definition given above, it follows that there is a well definedand only one value for the sequence identity between two comparedsequences which value corresponds to the value obtained for the best oroptimal alignment.

In the BLAST N or BLAST P “BLAST 2 sequence”, software which isavailable in the web site http://www.ncbi.nlm.nih.gov/gorf/b12.html, andhabitually used by the inventors and in general by the skilled man forcomparing and determining the identity between two sequences, gap costwhich depends on the sequence length to be compared is directly selectedby the software (i.e. 11.2 for substitution matrix BLOSUM-62 forlength>85).

In the present description, PWD circovirus will be understood asdesignating the circoviruses associated with piglet weight loss disease(PWD) of type A (PCVA) or type B (PCVB), defined below by their genomicsequence, as well as the circoviruses whose nucleic sequences arehomologous to the sequences of PWD circoviruses of type A or B, such asin particular the circoviruses corresponding to variants of the type Aor of the type B.

Complementary nucleotide sequence of a sequence of the invention isunderstood as meaning any DNA whose nucleotides are complementary tothose of the sequence of the invention, and whose orientation isreversed (antiparallel sequence).

Hybridization under conditions of stringency with a nucleotide sequenceaccording to the invention is understood as meaning a hybridizationunder conditions of temperature and ionic strength chosen in such a waythat they allow the maintenance of the hybridization between twofragments of complementary DNA.

By way of illustration, conditions of great stringency of thehybridization step with the aim of defining the nucleotide fragmentsdescribed above are advantageously the following.

The hybridization is carried out at a preferential temperature of 65° C.in the presence of SSC buffer, 1×SSC corresponding to 0.15 M NaCl and0.05 M Na citrate. The washing steps, for example, can be the following:

-   -   2×SSC, at ambient temperature followed by two washes with 2×SSC,        0.5% SDS at 65° C.; 2×0.5×SSC, 0.5% SDS; at 65° C. for 10        minutes each.

The conditions of intermediate stringency, using, for example, atemperature of 42° C. in the presence of a 2×SSC buffer, or of lessstringency, for example a temperature of 37° C. in the presence of a2×SSC buffer, respectively require a globally less significantcomplementarity for the hybridization between the two sequences.

The stringent hybridization conditions described above for apolynucleotide with a size of approximately 350 bases will be adapted bythe person skilled in the art for oligonucleotides of greater or smallersize, according to the teaching of Sambrook et al., 1989.

Among the nucleotide sequences according to the invention, those arelikewise preferred which can be used as a primer or probe in methodsallowing the homologous sequences according to the invention to beobtained, these methods, such as the polymerase chain reaction (PCR),nucleic acid cloning and sequencing, being well known to the personskilled in the art.

Among said nucleotide sequences according to the invention, those areagain preferred which can be used as a primer or probe in methodsallowing the presence of PWD circovirus or one of its variants such asdefined below to be diagnosed.

The nucleotide sequences according to the invention capable ofmodulating, of inhibiting or of inducing the expression of PWDcircovirus gene, and/or capable of modulating the replication cycle ofPWD circovirus in the host cell and/or organism are likewise preferred.Replication cycle will be understood as designating the invasion and themultiplication of PWD circovirus, and its propagation from host cell tohost cell in the host organism.

Among said nucleotide sequences according to the invention, thosecorresponding to open reading frames, called ORF sequences, and codingfor polypeptides, such as, for example, the sequences SEQ ID No. 9(ORF1), SEQ ID No. 11 (ORF2) and SEQ ID No. 13 (ORF3) respectivelycorresponding to the nucleotide sequences between the positions 47 and985 determined with respect to the position of the nucleotides on thesequence SEQ ID No. 1, the positions 1723 and 1022 and the positions 658and 38 with respect to the position of the nucleotides on the sequenceSEQ ID No. 5 (represented according to the orientation 3′→5′), the endsbeing included, or alternatively the sequences SEQ ID No. 23 (ORF′1),SEQ ID No. 25 (ORF′2) and SEQ ID No. 27 (ORF′3), respectivelycorresponding to the sequences between the positions 51 and 995determined with respect to the position of the nucleotides on thesequence SEQ ID No. 15, the positions 1734 and 1033 and the positions670 and 357, the positions being determined with respect to the positionof the nucleotides on the sequence SEQ ID No. 19 (represented accordingto the orientation 3′→5′), the ends being included, are finallypreferred.

The nucleotide sequence fragments according to the invention can beobtained, for example, by specific amplification, such as PCR, or afterdigestion with appropriate restriction enzymes of nucleotide sequencesaccording to the invention, these methods in particular being describedin the work of Sambrook et al., 1989. Said representative fragments canlikewise be obtained by chemical synthesis when their size is not verylarge and according to methods well known to persons skilled in the art.

Modified nucleotide sequence will be understood as meaning anynucleotide sequence obtained by mutagenesis according to techniques wellknown to the person skilled in the art, and containing modificationswith respect to the normal sequences according to the invention, forexample mutations in the regulatory and/or promoter sequences ofpolypeptide expression, especially leading to a modification of the rateof expression of said polypeptide or to a modulation of the replicativecycle.

Modified nucleotide sequence will likewise be understood as meaning anynucleotide sequence coding for a modified polypeptide such as definedbelow.

The present invention relates to nucleotide sequences of PWD circovirusaccording to the invention, characterized in that they are selected fromthe sequences SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 23,SEQ ID No. 25, SEQ ID No. 27 or one of their fragments.

The invention likewise relates to nucleotide sequences characterized inthat they comprise a nucleotide sequence selected from:

-   -   a) a nucleotide sequence SEQ ID No. 9, SEQ ID No. 11, SEQ ID No.        13, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27 or one of their        fragments;    -   b) a nucleotide sequence of a specific fragment of a sequence        such as defined in a);    -   c) a homologous nucleotide sequence having at least 80% identity        with a sequence such as defined in a) or b);    -   d) a complementary nucleotide sequence or sequence of RNA        corresponding to a sequence such as defined in a), b) or c); and    -   e) a nucleotide sequence modified by a sequence such as defined        in a), b), c) or d).

As far as homology with the nucleotide sequences SEQ ID No. 9, SEQ IDNo. 11, SEQ ID No. 13, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27 orone of their fragments is concerned, the homologous, especiallyspecific, sequences having a percentage identity with one of thesequences SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 23, SEQID No. 25, SEQ ID No. 27 or one of their fragments of at least 80%,preferably 90% or 95%, are preferred. Said specific homologous sequencescan comprise, for example, the sequences corresponding to the sequencesORF1, ORF2, ORF3, ORF′1, ORF′2 and ORF′3 of PWD circovirus variants oftype A or of type B. In the same manner, these specific homologoussequences can correspond to variations linked to mutations withinstrains of PWD circovirus of type A or of type B and especiallycorrespond to truncations, substitutions, deletions and/or additions ofat least one nucleotide.

Among nucleotide sequences according to the invention, the sequence SEQID No. 23 which has a homology having more than 80% identity with thesequence SEQ ID No. 9, as well as the sequence SEQ ID No. 25, areespecially preferred.

Preferably, the invention relates to the nucleotide sequences accordingto the invention, characterized in that they comprise a nucleotidesequence selected from the following sequences:

a) SEQ ID No. 33 170 5′ TGTGGCGA 3′; b) SEQ ID No. 34 450 5′ AGTTTCCT3′; c) SEQ ID No. 35 1026 5′ TCATTTAGAGGGTCTTTCAG 3′; d) SEQ ID No. 361074 5′ GTCAACCT 3′; e) SEQ ID No. 37 1101 5′ GTGGTTGC 3′; f) SEQ ID No.38 1123 5′ AGCCCAGG 3′; g) SEQ ID No. 39 1192 5′ TTGGCTGG 3′; h) SEQ IDNo. 40 1218 5′ TCTAGCTCTGGT 3′; i) SEQ ID No. 41 1501 5′ ATCTCAGCTCGT3′; j) SEQ ID No. 42 1536 5′ TGTCCTCCTCTT 3′; k) SEQ ID No. 43 15635′ TCTCTAGA 3′; l) SEQ ID No. 44 1623 5′ TGTACCAA 3′; m) SEQ ID No. 451686 5′ TCCGTCTT 3′;and their complementary sequences.

In the list of nucleotide sequences a)-m) above, the underlinednucleotides are mutated with respect to the two known sequences ofcircovirus which are nonpathogenic to pigs. The number preceding thenucleotide sequence represents the position of the first nucleotide ofsaid sequence in the sequence SEQ ID No. 1.

The invention comprises the polypeptides encoded by a nucleotidesequence according to the invention, preferably a polypeptide whosesequence is represented by a fragment, especially a specific fragment,of one of the six sequences of amino acids represented in FIG. 2, thesesix amino acid sequences corresponding to the polypeptides which can beencoded according to one of the three possible reading frames of thesequence SEQ ID No. 1 or of the sequence SEQ ID No. 5, or a polypeptidewhose sequence is represented by a fragment, especially a specificfragment, of one of the six sequences of amino acids shown in FIG. 8,these six sequences of amino acids corresponding to the polypeptideswhich can be encoded according to one of the three possible readingframes of the sequence SEQ ID No. 15 or of the sequence SEQ ID No. 19.

The invention likewise relates to the polypeptides, characterized inthat they comprise a polypeptide selected from the amino acid sequencesSEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 24, SEQ ID No.26, SEQ ID No. 28 or one of their fragments.

Among the polypeptides according to the invention, the polypeptide ofamino acid sequence SEQ ID No. 24 which has a homology having more than80% identity with the sequence SEQ ID No. 10, as well as the polypeptideof sequence SEQ ID No. 26, are especially preferred.

The invention also relates to the polypeptides, characterized in thatthey comprise a polypeptide selected from:

-   -   a) a specific fragment of at least 5 amino acids of a        polypeptide of an amino acid sequence according to the        invention;    -   b) a polypeptide homologous to a polypeptide such as defined in        a);    -   c) a specific biologically active fragment of a polypeptide such        as defined in a) or b); and    -   d) a polypeptide modified by a polypeptide such as defined in        a), b) or c).

Among the polypeptides according to the invention, the polypeptides ofamino acid sequences SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31 and SEQID No. 32 are also preferred, these polypeptides being especiallycapable of specifically recognizing the antibodies produced duringinfection by the PWD circovirus of type B. These polypeptides thus haveepitopes specific for the PWD circovirus of type B and can thus be usedin particular in the diagnostic field or as immunogenic agent to conferprotection in pigs against infection by PWD circovirus, especially oftype B.

In the present description, the terms polypeptide, peptide and proteinare interchangeable.

It must be understood that the invention does not relate to thepolypeptides in natural form, that is to say that they are not taken intheir natural environment but that they can be isolated or obtained bypurification from natural sources, or else obtained by geneticrecombination, or alternatively by chemical synthesis and that they canthus contain unnatural amino acids, as will be described below.

Polypeptide fragment according to the invention is understood asdesignating a polypeptide containing at least 5 consecutive amino acids,preferably 10 consecutive amino acids or 15 consecutive amino acids.

In the present invention, specific polypeptide fragment is understood asdesignating the consecutive polypeptide fragment encoded by a specificfragment nucleotide sequence according to the invention.

Homologous polypeptide will be understood as designating thepolypeptides having, with respect to the natural polypeptide, certainmodifications such as, in particular, a deletion, addition orsubstitution of at least one amino acid, a truncation, a prolongation, achimeric fusion, and/or a mutation. Among the homologous polypeptides,those are preferred whose amino acid sequence has at least 80%,preferably 90%, homology with the sequences of amino acids ofpolypeptides according to the invention.

Specific homologous polypeptide will be understood as designating thehomologous polypeptides such as defined above and having a specificfragment of polypeptide according to the invention.

In the case of a substitution, one or more consecutive or nonconsecutiveamino acids are replaced by “equivalent” amino acids. The expression“equivalent” amino acid is directed here at designating any amino acidcapable of being substituted by one of the amino acids of the basestructure without, however, essentially modifying the biologicalactivities of the corresponding peptides and such that they will bedefined by the following.

These equivalent amino acids can be determined either by depending ontheir structural homology with the amino acids which they substitute, oron results of comparative tests of biological activity between thedifferent polypeptides, which are capable of being carried out.

By way of example, the possibilities of substitutions capable of beingcarried out without resulting in an extensive modification of thebiological activity of the corresponding modified polypeptides will bementioned, the replacement, for example, of leucine by valine orisoleucine, of aspartic acid by glutamic acid, of glutamine byasparagine, of arginine by lysine etc., the reverse substitutionsnaturally being envisageable under the same conditions.

The specific homologous polypeptides likewise correspond to polypeptidesencoded by the specific homologous nucleotide sequences such as definedabove and thus comprise in the present definition the polypeptides whichare mutated or correspond to variants which can exist in PWD circovirus,and which especially correspond to truncations, substitutions, deletionsand/or additions of at least one amino acid residue.

Specific biologically active fragment of a polypeptide according to theinvention will be understood in particular as designating a specificpolypeptide fragment, such as defined above, having at least one of thecharacteristics of polypeptides according to the invention, especiallyin that it is:

-   -   capable of inducing an immunogenic reaction directed against a        PWD circovirus; and/or    -   capable of being recognized by a specific antibody of a        polypeptide according to the invention; and/or    -   capable of linking to a polypeptide or to a nucleotide sequence        of PWD circovirus; and/or    -   capable of exerting a physiological activity, even partial, such        as, for example, a dissemination or structural (capsid)        activity; and/or    -   capable of modulating, of inducing or of inhibiting the        expression of PWD circovirus gene or one of its variants, and/or        capable of modulating the replication cycle of PWD circovirus in        the cell and/or the host organism.

The polypeptide fragments according to the invention can correspond toisolated or purified fragments naturally present in a PWD circovirus orcorrespond to fragments which can be obtained by cleavage of saidpolypeptide by a proteolytic enzyme, such as trypsin or chymotrypsin orcollagenase, or by a chemical reagent, such as cyanogen bromide (CNBr)or alternatively by placing said polypeptide in a very acidicenvironment, for example at pH 2.5. Such polypeptide fragments canlikewise just as easily be prepared by chemical synthesis, from hoststransformed by an expression vector according to the inventioncontaining a nucleic acid allowing the expression of said fragments,placed under the control of appropriate regulation and/or expressionelements.

“Modified polypeptide” of a polypeptide according to the invention isunderstood as designating a polypeptide obtained by geneticrecombination or by chemical synthesis as will be described below,having at least one modification with respect to the normal sequence.These modifications will especially be able to bear on amino acids atthe origin of a specificity, of pathogenicity and/or of virulence, or atthe origin of the structural conformation, and of the capacity ofmembrane insertion of the polypeptide according to the invention. Itwill thus be possible to create polypeptides of equivalent, increased ordecreased activity, and of equivalent, narrower, or wider specificity.Among the modified polypeptides, it is necessary to mention thepolypeptides in which up to 5 amino acids can be modified, truncated atthe N- or C-terminal end, or even deleted or added.

As is indicated, the modifications of the polypeptide will especiallyhave as objective:

-   -   to render it capable of modulating, of inhibiting or of inducing        the expression of PWD circovirus gene and/or capable of        modulating the replication cycle of PWD circovirus in the cell        and/or the host organism,    -   of allowing its incorporation into vaccine compositions,    -   of modifying its bioavailability as a compound for therapeutic        use.

The methods allowing said modulations on eukaryotic or prokaryotic cellsto be demonstrated are well known to the person skilled in the art. Itis likewise well understood that it will be possible to use thenucleotide sequences coding for said modified polypeptides for saidmodulations, for example through vectors according to the invention anddescribed below, in order, for example, to prevent or to treat thepathologies linked to the infection.

The preceding modified polypeptides can be obtained by usingcombinatorial chemistry, in which it is possible to systematically varyparts of the polypeptide before testing them on models, cell cultures ormicroorganisms for example, to select the compounds which are mostactive or have the properties sought.

Chemical synthesis likewise has the advantage of being able to use:

-   -   unnatural amino acids, or    -   nonpeptide bonds.

Thus, in order to improve the duration of life of the polypeptidesaccording to the invention, it may be of interest to use unnatural aminoacids, for example in D form, or else amino acid analogs, especiallysulfur-containing forms, for example.

Finally, it will be possible to integrate the structure of thepolypeptides according to the invention, its specific or modifiedhomologous forms, into chemical structures of polypeptide type orothers. Thus, it may be of interest to provide at the N- and C-terminalends compounds not recognized by the proteases.

The nucleotide sequences coding for a polypeptide according to theinvention are likewise part of the invention.

The invention likewise relates to nucleotide sequences utilizable as aprimer or probe, characterized in that said sequences are selected fromthe nucleotide sequences according to the invention.

Among the pairs of nucleotide sequences utilizable as a pair of primersaccording to the invention, the pairs of primers selected from thefollowing pairs are preferred:

a) SEQ ID No. 46 5′ GTG TGC TCG ACA TTG GTG TG 3′, and SEQ ID No. 475′ TGG AAT GTT AAC GAG CTG AG 3′; b) SEQ ID No. 46 5′ GTG TGC TCG ACATTG GTG TG 3′, and SEQ ID No. 48 5′ CTC GCA GCC ATC TTG GAA TG 3′; c)SEQ ID No. 49 5′ CGC GCG TAA TAC GAC TCA CT 3′, and SEQ ID No. 46 5′ GTGTGC TCG ACA TTG GTG TG 3′; d) SEQ ID No. 49 5′ CGC GCG TAA TAC GAC TCACT 3′, and SEQ ID No. 48 5′ CTC GCA GCC ATC TTG GAA TG 3′; and e) SEQ IDNo. 50 5′ CCT GTC TAC TGC TGT GAG TAC CTT GT 3′, and SEQ ID No. 515′ GCA GTA GAC AGG TCA CTC CGT TGT CC 3′.

The cloning and the sequencing of the PWD circovirus, type A and B, hasallowed it to be identified, after comparative analysis with thenucleotide sequences of other porcine circoviruses, that, among thesequences of fragments of these nucleic acids, were those which arestrictly specific to the PWD circovirus of type A, of type B or of typeA and B, and those which correspond to a consensus sequence of porcinecircoviruses other than the PWD circoviruses of type A and/or B.

There is likewise a great need for nucleotide sequences utilizable as aprimer or probe specific to the whole of the other known andnonpathogenic porcine circoviruses.

Said consensus nucleotide sequences specific to all circoviruses, otherthan PWD circovirus of type A and B, are easily identifiable from FIG. 3and the sequence SEQ ID No. 15, and are part of the invention.

Among said consensus nucleotide sequences, that which is characterizedin that it is part of the following pair of primers is preferred:

a) SEQ ID No. 46 5′ GTG TGC TCG ACA TTG GTG TG 3′, and SEQ ID No. 525′ TGG AAT GTT AAC TAC CTC AA 3′.

The invention likewise comprises a nucleotide sequence according to theinvention, characterized in that said sequence is a specific consensussequence of porcine circovirus other than PWD circovirus of type B andin that it is one of the primers of the following pairs of primers:

a) SEQ ID No. 53 5′ GGC GGC GCC ATC TGT AAC GGT TT 3′, and SEQ ID No. 545′ GAT GGC GCC GAA AGA CGG GTA TC 3′.

It is well understood that the present invention likewise relates tospecific polypeptides of known porcine circoviruses other than PWDcircovirus, encoded by said consensus nucleotide sequences, capable ofbeing obtained by purification from natural polypeptides, by geneticrecombination or by chemical synthesis by procedures well known to theperson skilled in the art and such as described in particular below. Inthe same manner, the labeled or unlabeled mono- or polyclonal antibodiesdirected against said specific polypeptides encoded by said consensusnucleotide sequences are also part of the invention.

It will be possible to use said consensus nucleotide sequences, saidcorresponding polypeptides as well as said antibodies directed againstsaid polypeptides in procedures or sets for detection and/oridentification such as described below, in place of or in addition tonucleotide sequences, polypeptides or antibodies according to theinvention, specific to PWD circovirus type A and/or B.

These protocols have been improved for the differential detection of thecircular monomeric forms of specific replicative forms of the virion orof the DNA in replication and the dimeric forms found in so-calledin-tandem molecular constructs.

The invention additionally relates to the use of a nucleotide sequenceaccording to the invention as a primer or probe for the detection and/orthe amplification of nucleic acid sequences.

The nucleotide sequences according to the invention can thus be used toamplify nucleotide sequences, especially by the PCR technique(polymerase chain reaction) (Erlich, 1989; Innis et al., 1990; Rolfs etal., 1991; and White et al., 1997).

These oligodeoxyribonucleotide or oligoribonucleotide primersadvantageously have a length of at least 8 nucleotides, preferably of atleast 12 nucleotides, and even more preferentially at least 20nucleotides.

Other amplification techniques of the target nucleic acid can beadvantageously employed as alternatives to PCR.

The nucleotide sequences of the invention, in particular the primersaccording to the invention, can likewise be employed in other proceduresof amplification of a target nucleic acid, such as:

-   -   the TAS technique (Transcription-based Amplification System),        described by Kwoh et al. in 1989;    -   the 3SR technique (Self-Sustained Sequence Replication),        described by Guatelli et al. in 1990;    -   the NASBA technique (Nucleic Acid Sequence Based Amplification),        described by Kievitis et al. in 1991;    -   the SDA technique (Strand Displacement Amplification) (Walker et        al., 1992);    -   the TMA technique (Transcription Mediated Amplification).

The polynucleotides of the invention can also be employed in techniquesof amplification or of modification of the nucleic acid serving as aprobe, such as:

-   -   the LCR technique (Ligase Chain Reaction), described by        Landegren et al. in 1988 and improved by Barany et al. in 1991,        which employs a thermostable ligase;    -   the RCR technique (Repair Chain Reaction), described by Segev in        1992;    -   the CPR technique (Cycling Probe Reaction), described by Duck et        al. in 1990;    -   the amplification technique with Q-beta replicase, described by        Miele et al. in 1983 and especially improved by Chu et al. in        1986, Lizardi et al. in 1988, then by Burg et al. as well as by        Stone et al. in 1996.

In the case where the target polynucleotide to be detected is possiblyan RNA, for example an mRNA, it will be possible to use, prior to theemployment of an amplification reaction with the aid of at least oneprimer according to the invention or to the employment of a detectionprocedure with the aid of at least one probe of the invention, an enzymeof reverse transcriptase type in order to obtain a cDNA from the RNAcontained in the biological sample. The cDNA obtained will thus serve asa target for the primer(s) or the probe(s) employed in the amplificationor detection procedure according to the invention.

The detection probe will be chosen in such a manner that it hybridizeswith the target sequence or the amplicon generated from the targetsequence. By way of sequence, such a probe will advantageously have asequence of at least 12 nucleotides, in particular of at least 20nucleotides, and preferably of at least 100 nucleotides.

The invention also comprises the nucleotide sequences utilizable as aprobe or primer according to the invention, characterized in that theyare labeled with a radioactive compound or with a nonradioactivecompound.

The unlabeled nucleotide sequences can be used directly as probes orprimers, although the sequences are generally labeled with a radioactiveelement (³²P, ³⁵S, ³H, ¹²⁵I) or with a nonradioactive molecule (biotin,acetylaminofluorene, digoxigenin, 5-bromodeoxyuridine, fluorescein) toobtain probes which are utilizable for numerous applications.

Examples of nonradioactive labeling of nucleotide sequences aredescribed, for example, in French Patent No. 78.10975 or by Urdea et al.or by Sanchez-Pescador et al. in 1988.

In the latter case, it will also be possible to use one of the labelingmethods described in patents FR-2 422 956 and FR-2 518 755.

The hybridization technique can be carried out in various manners(Matthews et al., 1988). The most general method consists inimmobilizing the nucleic acid extract of cells on a support (such asnitrocellulose, nylon, polystyrene) and in incubating, underwell-defined conditions, the immobilized target nucleic acid with theprobe. After hybridization, the excess of probe is eliminated and thehybrid molecules formed are detected by the appropriate method(measurement of the radioactivity, of the fluorescence or of theenzymatic activity linked to the probe).

The invention likewise comprises the nucleotide sequences according tothe invention, characterized in that they are immobilized on a support,covalently or noncovalently.

According to another advantageous mode of employing nucleotide sequencesaccording to the invention, the latter can be used immobilized on asupport and can thus serve to capture, by specific hybridization, thetarget nucleic acid obtained from the biological sample to be tested. Ifnecessary, the solid support is separated from the sample and thehybridization complex formed between said capture probe and the targetnucleic acid is then detected with the aid of a second probe, aso-called detection probe, labeled with an easily detectable element.

Another subject of the present invention is a vector for the cloningand/or expression of a sequence, characterized in that it contains anucleotide sequence according to the invention.

The vectors according to the invention, characterized in that theycontain the elements allowing the expression and/or the secretion ofsaid nucleotide sequences in a determined host cell, are likewise partof the invention.

The vector must then contain a promoter, signals of initiation andtermination of translation, as well as appropriate regions of regulationof transcription. It must be able to be maintained stably in the hostcell and can optionally have particular signals specifying the secretionof the translated protein. These different elements are chosen as afunction of the host cell used. To this end, the nucleotide sequencesaccording to the invention can be inserted into autonomous replicationvectors within the chosen host, or integrated vectors of the chosenhost.

Such vectors will be prepared according to the methods currently used bythe person skilled in the art, and it will be possible to introduce theclones resulting therefrom into an appropriate host by standard methods,such as, for example, lipofection, electroporation and thermal shock.

The vectors according to the invention are, for example, vectors ofplasmid or viral origin.

A preferred vector for the expression of polypeptides of the inventionis baculovirus.

The vector pBS KS in which is inserted the in-tandem DNA sequence of thePWD circovirus type A (or DFP) as deposited at the CNCM on 3 Jul. 1997,under the number I-1891, is likewise preferred.

These vectors are useful for transforming host cells in order to cloneor to express the nucleotide sequences of the invention.

The invention likewise comprises the host cells transformed by a vectoraccording to the invention.

These cells can be obtained by the introduction into host cells of anucleotide sequence inserted into a vector such as defined above, thenthe culturing of said cells under conditions allowing the replicationand/or expression of the transfected nucleotide sequence.

The host cell can be selected from prokaryotic or eukaryotic systems,such as, for example, bacterial cells (Olins and Lee, 1993), butlikewise yeast cells (Buckholz, 1993), as well as animal cells, inparticular the cultures of mammalian cells (Edwards and Aruffo, 1993),and especially Chinese hamster ovary (CHO) cells, but likewise the cellsof insects in which it is possible to use procedures employingbaculoviruses, for example (Luckow, 1993).

A preferred host cell for the expression of the proteins of theinvention is constituted by sf9 insect cells.

A more preferred host cell according to the invention is E. coli, suchas deposited at the CNCM on 3 Jul. 1997, under the number I-1891.

The invention likewise relates to animals comprising one of saidtransformed cells according to the invention.

The obtainment of transgenic animals according to the inventionoverexpres sing one or more of the genes of PWD circovirus or part ofthe genes will be preferably carried out in rats, mice or rabbitsaccording to methods well known to the person skilled in the art, suchas by viral or nonviral transfections. It will be possible to obtain thetransgenic animals overexpressing one or more of said genes bytransfection of multiple copies of said genes under the control of astrong promoter of ubiquitous nature, or selective for one type oftissue. It will likewise be possible to obtain the transgenic animals byhomologous recombination in embryonic cell strains, transfer of thesecell strains to embryos, selection of the affected chimeras at the levelof the reproductive lines, and growth of said chimeras.

The transformed cells as well as the transgenic animals according to theinvention are utilizable in procedures for preparation of recombinantpolypeptides.

It is today possible to produce recombinant polypeptides in relativelylarge quantity by genetic engineering using the cells transformed byexpression vectors according to the invention or using transgenicanimals according to the invention.

The procedures for preparation of a polypeptide of the invention inrecombinant form, characterized in that they employ a vector and/or acell transformed by a vector according to the invention and/or atransgenic animal comprising one of said transformed cells according tothe invention, are themselves comprised in the present invention.

Among said procedures for preparation of a polypeptide of the inventionin recombinant form, the preparation procedures employing a vector,and/or a cell transformed by said vector and/or a transgenic animalcomprising one of said transformed cells, containing a nucleotidesequence according to the invention coding for a polypeptide of PWDcircovirus, are preferred.

The recombinant polypeptides obtained as indicated above can just aswell be present in glycosylated form as in nonglycosylated form and canor cannot have the natural tertiary structure.

A preferred variant consists in producing a recombinant polypeptide usedto a “carrier” protein (chimeric protein). The advantage of this systemis that it allows a stabilization of and a decrease in the proteolysisof the recombinant product, an increase in the solubility in the courseof renaturation in vitro and/or a simplification of the purificationwhen the fusion partner has an affinity for a specific ligand.

More particularly, the invention relates to a procedure for preparationof a polypeptide of the invention comprising the following steps:

-   -   a) culture of transformed cells under conditions allowing the        expression of a recombinant polypeptide of nucleotide sequence        according to the invention;    -   b) if need be, recovery of said recombinant polypeptide.

When the procedure for preparation of a polypeptide of the inventionemploys a transgenic animal according to the invention, the recombinantpolypeptide is then extracted from said animal.

The invention also relates to a polypeptide which is capable of beingobtained by a procedure of the invention such as described previously.

The invention also comprises a procedure for preparation of a syntheticpolypeptide, characterized in that it uses a sequence of amino acids ofpolypeptides according to the invention.

The invention likewise relates to a synthetic polypeptide obtained by aprocedure according to the invention.

The polypeptides according to the invention can likewise be prepared bytechniques which are conventional in the field of the synthesis ofpeptides. This synthesis can be carried out in homogeneous solution orin solid phase.

For example, recourse can be made to the technique of synthesis inhomogeneous solution described by Houben-Weyl in 1974.

This method of synthesis consists in successively condensing, two bytwo, the successive amino acids in the order required, or in condensingamino acids and fragments formed previously and already containingseveral amino acids in the appropriate order, or alternatively severalfragments previously prepared in this way, it being understood that itwill be necessary to protect beforehand all the reactive functionscarried by these amino acids or fragments, with the exception of aminefunctions of one and carboxyls of the other or vice-versa, which mustnormally be involved in the formation of peptide bonds, especially afteractivation of the carboxyl function, according to the methods well knownin the synthesis of peptides.

According to another preferred technique of the invention, recourse willbe made to the technique described by Merrifield.

To make a peptide chain according to the Merrifield procedure, recourseis made to a very porous polymeric resin, on which is immobilized thefirst C-terminal amino acid of the chain. This amino acid is immobilizedon a resin through its carboxyl group and its amine function isprotected. The amino acids which are going to form the peptide chain arethus immobilized, one after the other, on the amino group, which isdeprotected beforehand each time, of the portion of the peptide chainalready formed, and which is attached to the resin. When the whole ofthe desired peptide chain has been formed, the protective groups of thedifferent amino acids forming the peptide chain are eliminated and thepeptide is detached from the resin with the aid of an acid.

The invention additionally relates to hybrid polypeptides having atleast one polypeptide according to the invention, and a sequence of apolypeptide capable of inducing an immune response in man or animals.

Advantageously, the antigenic determinant is such that it is capable ofinducing a humoral and/or cellular response.

It will be possible for such a determinant to comprise a polypeptideaccording to the invention in glycosylated form used with a view toobtaining immunogenic compositions capable of inducing the synthesis ofantibodies directed against multiple epitopes. Said polypeptides ortheir glycosylated fragments are likewise part of the invention.

These hybrid molecules can be formed, in part, of a polypeptide carriermolecule or of fragments thereof according to the invention, associatedwith a possibly immunogenic part, in particular an epitope of thediphtheria toxin, the tetanus toxin, a surface antigen of the hepatitisB virus (patent FR 79 21811), the VP1 antigen of the poliomyelitis virusor any other viral or bacterial toxin or antigen.

The procedures for synthesis of hybrid molecules encompass the methodsused in genetic engineering for constructing hybrid nucleotide sequencescoding for the polypeptide sequences sought. It will be possible, forexample, to refer advantageously to the technique for obtainment ofgenes coding for fusion proteins described by Minton in 1984.

Said hybrid nucleotide sequences coding for a hybrid polypeptide as wellas the hybrid polypeptides according to the invention characterized inthat they are recombinant polypeptides obtained by the expression ofsaid hybrid nucleotide sequences are likewise part of the invention.

The invention likewise comprises the vectors characterized in that theycontain one of said hybrid nucleotide sequences. The host cellstransformed by said vectors, the transgenic animals comprising one ofsaid transformed cells as well as the procedures for preparation ofrecombinant polypeptides using said vectors, said transformed cellsand/or said transgenic animals are, of course, likewise part of theinvention.

The polypeptides according to the invention, the antibodies according tothe invention described below and the nucleotide sequences according tothe invention can advantageously be employed in procedures for thedetection and/or identification of PWD circovirus, or of porcinecircovirus other than a PWD circovirus, in a biological sample(biological tissue or fluid) capable of containing them. Theseprocedures, according to the specificity of the polypeptides, theantibodies and the nucleotide sequences according to the invention whichwill be used, will in particular be able to detect and/or to identify aPWD circovirus or a porcine circovirus other than a PWD circovirus orother than the PWD circovirus of type B.

The polypeptides according to the invention can advantageously beemployed in a procedure for the detection and/or the identification ofPWD circovirus of type A, of type B, of type A or B, or porcinecircovirus other than the PWD circovirus of type B, or of porcinecircovirus other than the PWD circovirus of type A or B, in a biologicalsample (biological tissue or fluid) capable of containing them,characterized in that it comprises the following steps:

-   -   a) contacting of this biological sample with a polypeptide or        one of its fragments according to the invention (under        conditions allowing an immunological reaction between said        polypeptide and the antibodies possibly present in the        biological sample);    -   b) demonstration of the antigen-antibody complexes possibly        formed.

In the present description, PWD circovirus, except if a particularmention is indicated, will be understood as designating a PWD circovirusof type A or of type B, and porcine circovirus other than PWD, except ifa particular mention is indicated, will be understood as designating aporcine circovirus other than a PWD circovirus of type A and B.

Preferably, the biological sample is formed by a fluid, for example apig serum, whole blood or biopsies.

Any conventional procedure can be employed for carrying out such adetection of the antigen-antibody complexes possibly formed.

By way of example, a preferred method brings into play immunoenzymaticprocesses according to the ELISA technique, by immunofluorescence, orradioimmunological processes (RIA) or their equivalent.

Thus, the invention likewise relates to the polypeptides according tothe invention, labeled with the aid of an adequate label such as of theenzymatic, fluorescent or radioactive type.

Such methods comprise, for example, the following steps:

-   -   deposition of determined quantities of a polypeptide composition        according to the invention in the wells of a microtiter plate,    -   introduction into said wells of increasing dilutions of serum,        or of a biological sample other than that defined previously,        having to be analyzed,    -   incubation of the microplate,    -   introduction into the wells of the microtiter plate of labeled        antibodies directed against pig immunoglobulins, the labeling of        these antibodies having been carried out with the aid of an        enzyme selected from those which are capable of hydrolyzing a        substrate by modifying the absorption of the radiation of the        latter, at least at a determined wavelength, for example at 550        nm,    -   detection, by comparison with a control test, of the quantity of        hydrolyzed substrate.

The invention likewise relates to a kit or set for the detection and/oridentification of PWD circovirus, of porcine circovirus other than a PWDcircovirus or of porcine circovirus other than the PWD circovirus oftype B, characterized in that it comprises the following elements:

-   -   a polypeptide according to the invention,    -   if need be, the reagents for the formation of the medium        favorable to the immunological or specific reaction,    -   if need be, the reagents allowing the detection of the        antigen-antibody complexes produced by the immunological        reaction between the polypeptide(s) of the invention and the        antibodies possibly present in the biological sample, these        reagents likewise being able to carry a label, or to be        recognized in their turn by a labeled reagent, more particularly        in the case where the polypeptide according to the invention is        not labeled,    -   if need be, a biological reference sample (negative control)        devoid of antibodies recognized by a polypeptide according to        the invention,    -   if need be, a biological reference sample (positive control)        containing a predetermined quantity of antibodies recognized by        a polypeptide according to the invention.

The polypeptides according to the invention allow monoclonal orpolyclonal antibodies to be prepared which are characterized in thatthey specifically recognize the polypeptides according to the invention.It will advantageously be possible to prepare the monoclonal antibodiesfrom hybridomas according to the technique described by Kohler andMilstein in 1975. It will be possible to prepare the polyclonalantibodies, for example, by immunization of an animal, in particular amouse, with a polypeptide or a DNA, according to the invention,associated with an adjuvant of the immune response, and thenpurification of the specific antibodies contained in the serum of theimmunized animals on an affinity column on which the polypeptide whichhas served as an antigen has previously been immobilized. The polyclonalantibodies according to the invention can also be prepared bypurification, on an affinity column on which a polypeptide according tothe invention has previously been immobilized, of the antibodiescontained in the serum of pigs infected by a PWD circovirus.

The invention likewise relates to mono- or polyclonal antibodies ortheir fragments, or chimeric antibodies, characterized in that they arecapable of specifically recognizing a polypeptide according to theinvention.

It will likewise be possible for the antibodies of the invention to belabeled in the same manner as described previously for the nucleicprobes of the invention, such as a labeling of enzymatic, fluorescent orradioactive type.

The invention is additionally directed at a procedure for the detectionand/or identification of PWD circovirus, of porcine circovirus otherthan a PWD circovirus, or other than the PWD circovirus of type B, in abiological sample, characterized in that it comprises the followingsteps:

-   -   a) contacting of the biological sample (biological tissue or        fluid) with a mono- or polyclonal antibody according to the        invention (under conditions allowing an immunological reaction        between said antibodies and the polypeptides of PWD circovirus,        of porcine circovirus other than a PWD circovirus, of porcine        circovirus other than the PWD circovirus of type B, possibly        present in the biological sample);    -   b) demonstration of the antigen-antibody complex possibly        formed.

Likewise within the scope of the invention is a kit or set for thedetection and/or the identification of PWD circovirus, of porcinecircovirus other than a PWD circovirus or of porcine circovirus otherthan the PWD circovirus of type B, characterized in that it comprisesthe following components:

-   -   a polyclonal or monoclonal antibody according to the invention,        if need be labeled;    -   if need be, a reagent for the formation of the medium favorable        to the carrying out of the immunological reaction;    -   if need be, a reagent allowing the detection of the        antigen-antibody complexes produced by the immunological        reaction, this reagent likewise being able to carry a label, or        being capable of being recognized in its turn by a labeled        reagent, more particularly in the case where said monoclonal or        polyclonal antibody is not labeled;    -   if need be, reagents for carrying out the lysis of cells of the        sample tested.

The present invention likewise relates to a procedure for the detectionand/or the identification of PWD, of porcine circovirus other than a PWDcircovirus or of porcine circovirus other than the PWD circovirus oftype B, in a biological sample, characterized in that it employs anucleotide sequence according to the invention.

More particularly, the invention relates to a procedure for thedetection and/or the identification of PWD circovirus, of porcinecircovirus other than a PWD circovirus or of porcine circovirus otherthan the PWD circovirus of type B, in a biological sample, characterizedin that it contains the following steps:

-   -   a) if need be, isolation of the DNA from the biological sample        to be analyzed;    -   b) specific amplification of the DNA of the sample with the aid        of at least one primer, or a pair of primers, according to the        invention;    -   c) demonstration of the amplification products.

These can be detected, for example, by the technique of molecularhybridization utilizing a nucleic probe according to the invention. Thisprobe will advantageously be labeled with a nonradioactive (cold probe)or radioactive element.

For the purposes of the present invention, “DNA of the biologicalsample” or “DNA contained in the biological sample” will be understoodas meaning either the DNA present in the biological sample considered,or possibly the cDNA obtained after the action of an enzyme of reversetranscriptase type on the RNA present in said biological sample.

Another aim of the present invention consists in a procedure accordingto the invention, characterized in that it comprises the followingsteps:

-   -   a) contacting of a nucleotide probe according to the invention        with a biological sample, the DNA contained in the biological        sample having, if need be, previously been made accessible to        hybridization under conditions allowing the hybridization of the        probe with the DNA of the sample;    -   b) demonstration of the hybrid formed between the nucleotide        probe and the DNA of the biological sample.

The present invention also relates to a procedure according to theinvention, characterized in that it comprises the following steps:

-   -   a) contacting of a nucleotide probe immobilized on a support        according to the invention with a biological sample, the DNA of        the sample having, if need be, previously been made accessible        to hybridization, under conditions allowing the hybridization of        the probe with the DNA of the sample;    -   b) contacting of the hybrid formed between the nucleotide probe        immobilized on a support and the DNA contained in the biological        sample, if need be after elimination of the DNA of the        biological sample which has not hybridized with the probe, with        a nucleotide probe labeled according to the invention;    -   c) demonstration of the novel hybrid formed in step b).

According to an advantageous embodiment of the procedure for detectionand/or identification defined previously, this is characterized in that,prior to step a), the DNA of the biological sample is first amplifiedwith the aid of at least one primer according to the invention.

The invention is additionally directed at a kit or set for the detectionand/or the identification of PWD circovirus, of porcine circovirus otherthan the PWD circovirus or of porcine circovirus other than the PWDcircovirus of type B, characterized in that it comprises the followingelements:

-   -   a) a nucleotide probe according to the invention;    -   b) if need be, the reagents necessary for the carrying out of a        hybridization reaction;    -   c) if need be, at least one primer according to the invention as        well as the reagents necessary for an amplification reaction of        the DNA.

The invention likewise relates to a kit or set for the detection and/orthe identification of PWD circovirus, of porcine circovirus other than aPWD circovirus or of porcine circovirus other than the PWD circovirus oftype B, characterized in that it comprises the following components:

-   -   a) a nucleotide probe, called a capture probe, according to the        invention;    -   b) an oligonucleotide probe, called a revealing probe, according        to the invention,    -   c) if need be, at least one primer according to the invention,        as well as the reagents necessary for an amplification reaction        of the DNA.

The invention also relates to a kit or set for the detection and/oridentification of PWD circovirus, of porcine circovirus other than a PWDcircovirus or of porcine circovirus other than the PWD circovirus oftype B, characterized in that it comprises the following elements:

-   -   a) at least one primer according to the invention;    -   b) if need be, the reagents necessary for carrying out a DNA        amplification reaction;    -   c) if need be, a component allowing the sequence of the        amplified fragment to be verified, more particularly an        oligonucleotide probe according to the invention.

The invention additionally relates to the use of a nucleotide sequenceaccording to the invention, of a polypeptide according to the invention,of an antibody according to the invention, of a cell according to theinvention, and/or of an animal transformed according to the invention,for the selection of an organic or inorganic compound capable ofmodulating, inducing or inhibiting the expression of genes, and/or ofmodifying the cellular replication of PWD circovirus or capable ofinducing or of inhibiting the pathologies linked to an infection by aPWD circovirus.

The invention likewise comprises a method of selection of compoundscapable of binding to a polypeptide or one of its fragments according tothe invention, capable of binding to a nucleotide sequence according tothe invention, or capable of recognizing an antibody according to theinvention, and/or capable of modulating, inducing or inhibiting theexpression of genes, and/or of modifying the cellular replication of PWDcircovirus or capable of inducing or inhibiting the pathologies linkedto an infection by a PWD circovirus, characterized in that it comprisesthe following steps:

-   -   a) contacting of said compound with said polypeptide, said        nucleotide sequence, or with a cell transformed according to the        invention and/or administration of said compound to an animal        transformed according to the invention;    -   b) determination of the capacity of said compound to bind to        said polypeptide or said nucleotide sequence, or to modulate,        induce or inhibit the expression of genes, or to modulate the        growth or the replication of PWD circovirus, or to induce or        inhibit in said transformed animal the pathologies linked to an        infection by PWD circovirus (designated activity of said        compound).

The compounds capable of being selected can be organic compounds such aspolypeptides or carbohydrates or any other organic or inorganiccompounds already known, or novel organic compounds elaborated bymolecular modeling techniques and obtained by chemical or biochemicalsynthesis, these techniques being known to the person skilled in theart.

It will be possible to use said selected compounds to modulate thecellular replication of PWD circovirus and thus to control infection bythis virus, the methods allowing said modulations to be determined beingwell known to the person skilled in the art.

This modulation can be carried out, for example, by an agent capable ofbinding to a protein and thus of inhibiting or of potentiating itsbiological activity, or capable of binding to an envelope protein of theexternal surface of said virus and of blocking the penetration of saidvirus into the host cell or of favoring the action of the immune systemof the infected organism directed against said virus. This modulationcan likewise be carried out by an agent capable of binding to anucleotide sequence of a DNA of said virus and of blocking, for example,the expression of a polypeptide whose biological or structural activityis necessary for the replication or for the proliferation of said virushost cells to host cells in the host animal.

The invention relates to the compounds capable of being selected by aselection method according to the invention.

The invention likewise relates to a pharmaceutical compositioncomprising a compound selected from the following compounds:

-   -   a) a nucleotide sequence according to the invention;    -   b) a polypeptide according to the invention;    -   c) a vector, a viral particle or a cell transformed according to        the invention;    -   d) an antibody according to the invention;    -   e) a compound capable of being selected by a selection method        according to the invention;        possibly in combination with a pharmaceutically acceptable        vehicle and, if need be, with one or more adjuvants of the        appropriate immunity.

The invention also relates to an immunogenic and/or vaccine composition,characterized in that it comprises a compound selected from thefollowing compounds:

-   -   a) a nucleotide sequence according to the invention;    -   b) a polypeptide according to the invention;    -   c) a vector or a viral particle according to the invention; and    -   d) a cell according to the invention.

In one embodiment, the vaccine composition according to the invention ischaracterized in that it comprises a mixture of at least two of saidcompounds a), b), c) and d) above and in that one of the two saidcompounds is related to the PWD circovirus of type A and the other isrelated to the PWD circovirus of type B.

In another embodiment of the invention, the vaccine composition ischaracterized in that it comprises at least one compound a), b), c), ord) above which is related to PWD circovirus of type B. In still anotherembodiment, the vaccine composition is characterized in that itcomprises at least one compound a), b), c), or d) above which is relatedto PWD circovirus of type B ORF′2.

A compound related to the PWD circovirus of type A or of type B isunderstood here as respectively designating a compound obtained from thegenomic sequence of the PWD circovirus of type A or of type B.

The invention is additionally aimed at an immunogenic and/or vaccinecomposition, characterized in that it comprises at least one of thefollowing compounds:

-   -   a nucleotide sequence SEQ ID No. 23, SEQ ID No. 25, or one of        their fragments or homologues;    -   a polypeptide of sequence SEQ ID No. 24, SEQ ID No. 26, or one        of their fragments, or a modification thereof;    -   a vector or a viral particle comprising a nucleotide sequence        SEQ ID No. 23, SEQ ID No. 25, or one of their fragments or        homologues;    -   a transformed cell capable of expressing a polypeptide of        sequence SEQ ID No. 24, SEQ ID No. 26, or one of their        fragments, or a modification thereof; or    -   a mixture of at least two of said compounds.

The invention also comprises an immunogenic and/or vaccine compositionaccording to the invention, characterized in that it comprises saidmixture of at least two of said compounds as a combination product forsimultaneous, separate or protracted use for the prevention or thetreatment of infection by a PWD circovirus, especially of type B.

In a preferred embodiment, the vaccine composition according to theinvention comprises the mixture of the following compounds:

-   -   a pcDNA3 plasmid containing a nucleic acid of sequence SEQ ID        No. 23;    -   a pcDNA3 plasmid containing a nucleic acid of sequence SEQ ID        No. 25;    -   a pcDNA3 plasmid containing a nucleic acid coding for the GM-CSF        protein;    -   a recombinant baculovirus containing a nucleic acid of sequence        SEQ ID No. 23;    -   a recombinant baculovirus containing a nucleic acid of sequence        SEQ ID No. 25; and    -   if need be, an adjuvant of the appropriate immunity, especially        the adjuvant AIF™.

The invention is likewise directed at a pharmaceutical compositionaccording to the invention, for the prevention or the treatment of aninfection by a PWD circovirus.

The invention is also directed at a pharmaceutical composition accordingto the invention for the prevention or the treatment of an infection bythe PWD circovirus of type B.

The invention likewise concerns the use of a composition according tothe invention, for the preparation of a medicament intended for theprevention or the treatment of infection by a PWD circovirus, preferablyby the PWD circovirus of type B.

Under another aspect, the invention relates to a vector, a viralparticle or a cell according to the invention, for the treatment and/orthe prevention of a disease by gene therapy.

Finally, the invention comprises the use of a vector, of a viralparticle or of a cell according to the invention for the preparation ofa medicament intended for the treatment and/or the prevention of adisease by gene therapy.

The polypeptides of the invention entering into the immunogenic orvaccine compositions according to the invention can be selected bytechniques known to the person skilled in the art such as, for example,depending on the capacity of said polypeptides to stimulate the T cells,which is translated, for example, by their proliferation or thesecretion of interleukins, and which leads to the production ofantibodies directed against said polypeptides.

In pigs, as in mice, in which a weight dose of the vaccine compositioncomparable to the dose used in man is administered, the antibodyreaction is tested by taking of the serum followed by a study of theformation of a complex between the antibodies present in the serum andthe antigen of the vaccine composition, according to the usualtechniques.

The pharmaceutical compositions according to the invention will containan effective quantity of the compounds of the invention, that is to sayin sufficient quantity of said compound(s) allowing the desired effectto be obtained, such as, for example, the modulation of the cellularreplication of PWD circovirus. The person skilled in the art will knowhow to determine this quantity, as a function, for example, of the ageand of the weight of the individual to be treated, of the state ofadvancement of the pathology, of the possible secondary effects and bymeans of a test of evaluation of the effects obtained on a populationrange, these tests being known in these fields of application.

According to the invention, said vaccine combinations will preferably becombined with a pharmaceutically acceptable vehicle and, if need be,with one or more adjuvants of the appropriate immunity.

Today, various types of vaccines are available for protecting animals orman against infectious diseases: attenuated living microorganisms (M.bovis—BCG for tuberculosis), inactivated microorganisms (influenzavirus), acellular extracts (Bordetella pertussis for whooping cough),recombined proteins (surface antigen of the hepatitis B virus),polysaccharides (pneumococcal). Vaccines prepared from syntheticpeptides or genetically modified microorganisms expressing heterologousantigens are in the course of experimentation. More recently still,recombined plasmid DNAs carrying genes coding for protective antigenshave been proposed as an alternative vaccine strategy. This type ofvaccination is carried out with a particular plasmid originating from aplasmid of E. coli which does not replicate in vivo and which codesuniquely for the vaccinating protein. Animals have been immunized bysimply injecting the naked plasmid DNA into the muscle. This techniqueleads to the expression of the vaccine protein in situ and to an immuneresponse of cellular type (CTL) and of humoral type (antibody). Thisdouble induction of the immune response is one of the principaladvantages of the vaccination technique with naked DNA.

The vaccine compositions comprising nucleotide sequences or vectors intowhich are inserted said sequences are especially described in theinternational application No. WO 90/11092 and likewise in theinternational application No. WO 95/11307.

The constitutive nucleotide sequence of the vaccine compositionaccording to the invention can be injected into the host after havingbeen coupled to compounds which favor the penetration of thispolynucleotide into the interior of the cell or its transport to thecell nucleus. The resultant conjugates can be encapsulated in polymericmicroparticles, as described in the international application No. WO94/27238 (Medisorb Technologies International).

According to another embodiment of the vaccine composition according tothe invention, the nucleotide sequence, preferably a DNA, is complexedwith DEAE-dextran (Pagano et al., 1967) or with nuclear proteins (Kanedaet al., 1989), with lipids (Felgner et al., 1987) or encapsulated inliposomes (Fraley et al., 1980) or else introduced in the form of a gelfacilitating its transfection into the cells (Midoux et al., 1993,Pastore et al., 1994). The polynucleotide or the vector according to theinvention can also be in suspension in a buffer solution or be combinedwith liposomes.

Advantageously, such a vaccine will be prepared according to thetechnique described by Tacson et al. or Huygen et al. in 1996 oralternatively according to the technique described by Davis et al. inthe international application No. WO 95/11307.

Such a vaccine can likewise be prepared in the form of a compositioncontaining a vector according to the invention, placed under the controlof regulation elements allowing its expression in man or animal. It willbe possible, for example, to use, by way of in vivo expression vector ofthe polypeptide antigen of interest, the plasmid pcDNA3 or the plasmidpcDNA1/neo, both marketed by Invitrogen (R&D Systems, Abingdon, UnitedKingdom). It is also possible to use the plasmid V1Jns.tPA, described byShiver et al. in 1995. Such a vaccine will advantageously comprise,apart from the recombinant vector, a saline solution, for example asodium chloride solution.

Pharmaceutically acceptable vehicle is understood as designating acompound or a combination of compounds entering into a pharmaceuticalcomposition or vaccine which does not provoke secondary reactions andwhich allows, for example, the facilitation of the administration of theactive compound, an increase in its duration of life and/or its efficacyin the body, an increase in its solubility in solution or alternativelyan improvement in its conservation. These pharmaceutically acceptablevehicles are well known and will be adapted by the person skilled in theart as a function of the nature and of the mode of administration of thechosen active compound.

As far as the vaccine formulations are concerned, these can compriseadjuvants of the appropriate immunity which are known to the personskilled in the art, such as, for example, aluminum hydroxide, arepresentative of the family of muramyl peptides such as one of thepeptide derivatives of N-acetyl muramyl, a bacterial lysate, oralternatively Freund's incomplete adjuvant.

These compounds can be administered by the systemic route, in particularby the intravenous route, by the intramuscular, intradermal orsubcutaneous route, or by the oral route. In a more preferred manner,the vaccine composition comprising polypeptides according to theinvention will be administered by the intramuscular route, through thefood or by nebulization several times, staggered over time.

Their administration modes, dosages and optimum pharmaceutical forms canbe determined according to the criteria generally taken into account inthe establishment of a treatment adapted to an animal such as, forexample, the age or the weight, the seriousness of its generalcondition, the tolerance to the treatment and the secondary effectsnoted. Preferably, the vaccine of the present invention is administeredin an amount that is protective against piglet weight loss disease.

For example, in the case of a vaccine according to the present inventioncomprising a polypeptide encoded by a nucleotide sequence of the genomeof PCV, or a homologue or fragment thereof, the polypeptide will beadministered one time or several times, spread out over time, directlyor by means of a transformed cell capable of expressing the polypeptide,in an amount of about 0.1 to 10 μg per kilogram weight of the animal,preferably about 0.2 to about 5 μg/kg, more preferably about 0.5 toabout 2 μg/kg for a dose.

The present invention likewise relates to the use of nucleotidesequences of PWD circovirus according to the invention for theconstruction of autoreplicative retroviral vectors and the therapeuticapplications of these, especially in the field of human gene therapy invivo.

The feasibility of gene therapy applied to man no longer needs to bedemonstrated and this relates to numerous therapeutic applications likegenetic diseases, infectious diseases and cancers. Numerous documents ofthe prior art describe the means of employing gene therapy, especiallythrough viral vectors. Generally speaking, the vectors are obtained bydeletion of at least some of the viral genes which are replaced by thegenes of therapeutic interest. Such vectors can be propagated in acomplementation line which supplies in trans the deleted viral functionsin order to generate a defective viral vector particle for replicationbut capable of infecting a host cell. To date, the retroviral vectorsare amongst the most widely used and their mode of infection is widelydescribed in the literature accessible to the person skilled in the art.

The principle of gene therapy is to deliver a functional gene, called agene of interest, of which the RNA or the corresponding protein willproduce the desired biochemical effect in the targeted cells or tissues.On the one hand, the insertion of genes allows the prolonged expressionof complex and unstable molecules such as RNAs or proteins which can beextremely difficult or even impossible to obtain or to administerdirectly. On the other hand, the controlled insertion of the desiredgene into the interior of targeted specific cells allows the expressionproduct to be regulated in defined tissues. For this, it is necessary tobe able to insert the desired therapeutic gene into the interior ofchosen cells and thus to have available a method of insertion capable ofspecifically targeting the cells or the tissues chosen.

Among the methods of insertion of genes, such as, for example,microinjection, especially the injection of naked plasmid DNA (Derse, D.et al., 1995, and Zhao, T. M. et al., 1996), electroporation, homologousrecombination, the use of viral particles, such as retroviruses, iswidespread. However, applied in vivo, the gene transfer systems ofrecombinant retroviral type at the same time have a weak infectiouspower (insufficient concentration of viral particles) and a lack ofspecificity with regard to chosen target cells.

The production of cell-specific viral vectors, having a tissue-specifictropism, and whose gene of interest can be translated adequately by thetarget cells, is realizable, for example, by fusing a specific ligand ofthe target host cells to the N-terminal part of a surface protein of theenvelope of PWD circovirus. It is possible to mention, for example, theconstruction of retroviral particles having the CD4 molecule on thesurface of the envelope so as to target the human cells infected by theHW virus (YOUNG, J. A. T. et al., Sciences 1990, 250, 1421-1423), viralparticles having a peptide hormone fused to an envelope protein tospecifically infect the cells expressing the corresponding receptor(KASAHARA, N. et al., Sciences 1994, 266, 1373-1376) or elsealternatively viral particles having a fused polypeptide capable ofimmobilizing on the receptor of the epidermal growth factor (EGF)(COSSET, F. L. et al., J. of Virology 1995, 69, 10, 6314-6322). Inanother approach, single-chain fragments of antibodies directed againstsurface antigens of the target cells are inserted by fusion with theN-terminal part of the envelope protein (VALSESIA-WITTMAN, S. et al., J.of Virology 1996, 70, 3, 2059-2064; TEARINA CHU, T. H. et al., J. ofVirology 1997, 71, 1, 720-725).

For the purposes of the present invention, a gene of interest in use inthe invention can be obtained from a eukaryotic or prokaryotic organismor from a virus by any conventional technique. It is, preferably,capable of producing an expression product having a therapeutic effectand it can be a product homologous to the cell host or, alternatively,heterologous. In the scope of the present invention, a gene of interestcan code for an (i) intracellular or (ii) membrane product present onthe surface of the host cell or (iii) secreted outside the host cell. Itcan therefore comprise appropriate additional elements such as, forexample, a sequence coding for a secretion signal. These signals areknown to the person skilled in the art.

In accordance with the aims pursued by the present invention, a gene ofinterest can code for a protein corresponding to all or part of a nativeprotein as found in nature. It can likewise be a chimeric protein, forexample arising from the fusion of polypeptides of various origins orfrom a mutant having improved and/or modified biological properties.Such a mutant can be obtained, by conventional biological techniques, bysubstitution, deletion and/or addition of one or more amino acidresidues.

It is very particularly preferred to employ a gene of therapeuticinterest coding for an expression product capable of inhibiting orretarding the establishment and/or the development of a genetic oracquired disease. A vector according to the invention is in particularintended for the prevention or for the treatment of cystic fibrosis, ofhemophilia A or B, of Duchenne's or Becker's myopathy, of cancer, ofAIDS and of other bacteria or infectious diseases due to a pathogenicorganism: virus, bacteria, parasite or prion. The genes of interestutilizable in the present invention are those which code, for example,for the following proteins:

-   -   a cytokine and especially an interleukin, an interferon, a        tissue necrosis factor and a growth factor and especially a        hematopoietic growth factor (G-CSF, GM-CSF),    -   a factor or cofactor involved in clotting and especially factor        VIII, von Willebrand's factor, antithrombin III, protein C,        thrombin and hirudin,    -   an enzyme or an enzyme inhibitor such as the inhibitors of viral        proteases,    -   an expression product of a suicide gene such as thymidine kinase        of the HSV virus (herpesvirus) of type 1,    -   an activator or an inhibitor of ion channels,    -   a protein of which the absence, the modification or the        deregulation of expression is responsible for a genetic disease,        such as the CFTR protein, dystrophin or minidystrophin, insulin,        ADA (adenosine diaminose), glucocerebrosidase and        phenylhydroxylase,    -   a protein capable of inhibiting the initiation or the        progression of cancers, such as the expression products of tumor        suppressor genes, for example the P53 and Rb genes,    -   a protein capable of stimulating an immune or an antibody        response, and    -   a protein capable of inhibiting a viral infection or its        development, for example the antigenic epitopes of the virus in        question or altered variants of viral proteins capable of        entering into competition with the native viral proteins.

The invention thus relates to the vectors characterized in that theycomprise a nucleotide sequence of PWD circovirus according to theinvention, and in that they additionally comprise a gene of interest.

The present invention likewise relates to viral particles generated fromsaid vector according to the invention. It additionally relates tomethods for the preparation of viral particles according to theinvention, characterized in that they employ a vector according to theinvention, including viral pseudoparticles (VLP, virus-like particles).

The invention likewise relates to animal cells transfected by a vectoraccording to the invention.

Likewise comprised in the invention are animal cells, especiallymammalian, infected by a viral particle according to the invention.

The present invention likewise relates to a vector, a viral particle ora cell according to the invention, for the treatment and/or theprevention of a genetic disease or of an acquired disease such as canceror an infectious disease. The invention is likewise directed at apharmaceutical composition comprising, by way of therapeutic orprophylactic agent, a vector or a cell according to the invention, incombination with a vehicle acceptable from a pharmaceutical point ofview.

Other characteristics and advantages of the invention appear in theexamples and the figures.

The invention is described in more detail in the following illustrativeexamples. Although the examples may represent only selected embodimentsof the invention, it should be understood that the following examplesare illustrative and not limiting.

Examples Example 1 Cloning, Sequencing and Characterization of the PWDCircovirus of Type A (PCVA)

1. Experimental Procedures

Experimental reproduction of the infection and its syndrome are provided(cf. FIG. 1).

A first test was carried out with pigs from a very well-kept farm, butaffected by piglet weight loss disease (PWD), likewise called fatalpiglet wasting (FPW). Tests carried out with SPF (specificpathogen-free) pigs showed a transfer of contaminant(s) findingexpression in a complex pathology combining hyperthermia, retardation ofgrowth, diarrhea and conjunctivitis. The PDRS (porcine dysgenic andrespiratory syndrome) virus, an infectious disease due to anarteriovirus) was rapidly isolated from breeding pigs and contact pigs.It should have been possible to attribute all the clinical signs to thepresence of the PDRS virus. However, two farm pigs presented signs ofFPW without the PDRS virus being isolated. The histological analyses andblood formulas, however, showed that these pigs were suffering from aninfectious process of viral origin.

In a second test, 8-week SPF pigs were inoculated by the intratrachealroute with organ homogenates of two farm pigs suffering from FPW. Theinoculated pigs exhibited hyperthermia 8 to 9 days post-infection, thentheir growth was retarded. Other SPF pigs, placed in contact, hadsimilar, attenuated signs 30 days after the initial experiment. Noseroconversion with respect to a European or Canadian strain of PDRSvirus was recorded in these animals.

A third test allowed the syndrome to be reproduced from samples takenfrom the pigs of the second test.

Conclusion

The syndrome is reproduced under the experimental conditions. It isdetermined by at least one infectious agent, which is transmittable bydirect contact. The clinical constants are a sometimes high hyperthermia(greater than or equal to 41.5° C.) which develops 8 to 10 days afterinfection. Retardation of the growth can be observed. The other signsare a reversal of the blood formula (reversal of thelymphocyte/polynuclear ratio from 70/30 to 30/70) and frequent lesionson the ganglia, especially those draining the respiratory apparatus(ganglionic hypertrophy, loss of structure with necrosis andinfiltration by mononucleated or plurinucleated giant cells).

2. Laboratory Studies

Various cell supports including primary pig kidney cells or cell lines,pig testicle cells, monkey kidney cells, pig lymphocytes, pig alveolarmacrophages and circulating blood monocytes were used to demonstrate thepossible presence of a virus. No cytopathic effect was demonstrated inthese cells. On the other hand, the use of a serum of a pig sick afterexperimental infection allowed an intracellular antigen to be revealedin the monocytes, the macrophages and approximately 10% of pig kidney(PK) cells infected with organ homogenates. This indirect revealing wascarried out kinetically at different culture times. It is evident fromthis that the antigen initially appears in the nucleus of the infectedcells before spreading into the cytoplasm. The successive passages incell culture did not allow the signal to be amplified.

Under electron microscopy on organ homogenates, spherical particleslabeled specifically by the serum of sick pigs, infected under theexperimental conditions, were visualized. The size of these particles isestimated at 20 nm.

After two passages of these organ homogenates over pig lymphocytes andthen three passages over pig kidney or testicle cells, a cytopathiceffect developed and was amplified. An adenovirus was visualized in theelectron microscope, which, under the experimental conditions, did notreproduce FPW (only a hyperthermia peak was noted 24 to 48 hours afterinfection, and then nothing more).

It has been possible to demonstrate DNA bands in certain samples of pigsinfected under the experimental conditions and having exhibited signs ofthe disease (results not shown). A certain connection exists between thesamples giving a positive result in cell culture and those having a DNAband.

Conclusion

At least two types of virus were demonstrated in the organ homogenatesfrom pigs suffering from FPW. One is an adenovirus, but by itself aloneit does not reproduce the disease. The other type of virus is acircovirus and is associated with FPW. This circovirus, of which twotypes have been isolated and sequenced, designated below PWD circovirustype A (or PCVA) and PWD circovirus of type B (or PCVB) have mutationswith respect to the known sequences of circovirus which arenonpathogenic for the pig.

3. Cloning and Sequencing of the DNA of the PWD Circovirus of Type A

Cloning and sequencing of the DNA of PHD circovirus Type A isaccomplished by extraction of the replicative form (RF) DNA, followed bycleavage by the Kpn I enzyme and amplification by a pair of primersflanking the Kpn I restriction site. The two strands of DNA aresequenced at least twice by the Sanger method.

The nucleic sequence of the strand of (+) polarity of the genome of thePWD circovirus of type A (or PCVA), strain FPW, is represented by thesequence SEQ ID No. 1 in the list of sequences, the nucleic acidsequence of the strand of (−) polarity of the genome of the PWDcircovirus of type A (or PCVA) being represented by the nucleic acidsequence 3′→5′ of FIG. 3 or by the sequence SEQ ID No. 5 (representedaccording to the orientation 5′→3′) in the list of sequences.

The amino acid sequences SEQ ID No. 10, SEQ ID No. 12 and SEQ ID No. 14of the list of sequences respectively represent the sequences ofproteins encoded by the nucleic sequences of the 3 open reading framesSEQ ID No. 9 (ORF1), corresponding to the REP protein, SEQ ID No. 11(ORF2) and SEQ ID No. 13 (ORF3), determined from the sequence SEQ ID No.1 of the strand of (+) polarity or of the nucleic sequence SEQ ID No. 5of the strand of (−) polarity of the genome of the PWD circovirus oftype A.

4. Comparison of the Nucleotide Sequences and Amino Acids of the PWDCircovirus of Type A (or Associated with PWD) which are Obtained withthe Corresponding Sequences of MEEHAN and MANKERTZ Circoviruses ofPorcine Cell Lines.

DNA sequences are analyzed using, DNASIS software.

Sequences of Oligonucleotides Used as Primers or Probes in the Detectionand/or Identification Procedures

1. Specific Detection of the PWD Circovirus of Type A:

SEQ ID No. 46 primer PCV 5: 5′ GTG TGC TCG ACA TTG GTG TG 3′; SEQ ID No.47 primer PCV 10: 5′ TGG AAT GTT AAC GAG CTG AG 3′;

2. Specific Detection of the Circovirus of the Cell Lines:

SEQ ID No. 46 primer PCF 5: 5′ GTG TGC TCG ACA TTG GTG TG 3′; SEQ ID No.52 primer MEE 1: 5′ TGG AAT GTT AAC TAC CTC AA 3′;

3. Differential Detection:

the pairs of primers used are those described, for example, in theparagraphs 1 and 2 above;

4. Detection of the Monomeric Circular Replicative Forms RF:

SEQ ID No. 46 primer PCV 5: 5′ GTG TGC TCG ACA TTG GTG TG 3′; SEQ ID No.48 primer PCV 6: 5′ CTC GCA GCC ATC TTG GAA TG 3′;

5. Detection of the Vectors Carrying the Dimers in Tandem:

Nar dimer: SEQ ID No. 49 primer KS 620: 5′ CGC GCG TAA TAC GAC TCA CT3′; SEQ ID No. 46 primer PCV 5: 5′ GTG TGC TCG ACA TTG GTG TG 3′; Kpndimer: SEQ ID No. 49 primer KS 620: 5′ CGC GCG TAA TAC GAC TCA CT 3′;SEQ ID No. 48 primer PCV 6: 5′CTC GCA GCC ATC TTG GAA TG 3′;

6. Differential Detection:

The pairs of primers used are those described, for example, inparagraphs 4 and 5 above.

The procedures using the pairs or primers described in paragraphs 4 and5 are of particular interest for differentially detecting the circularmonomeric forms of specific replicative forms of the virion or of theDNA in replication and the dimeric forms found in the so-calledin-tandem molecular constructs.

The in-tandem constructs of the viral genome (dimers) such as theconstructs used for the preparation of the pBS KS+tandem PCV Kpn Ivector, deposited at the CNCM under the number I-1891, 3 Jul. 1997 (E.coli transformed by said vector) are very interesting for their use inmethods of production of sufficient quantity of an inoculum formed ofDNA, intended for the virus production, this in the absence of asatisfactory virus production protocol in a cell system. These saidmethods of production using in-tandem constructs of the viral genomewill allow the virulence factors to be studied by mutation and by way ofconsequence will be able to be used for the production of a collectionof viruses carrying the mutations indicated in the construction ofvectors which will have the appropriate tropism and virulence. Thesevectors with autoreplicative structure have the sought gene transferproperties, especially for their applications in gene therapy, and invaccinology.

Western-Blot Analysis of Recombinant Proteins of the PWD Circovirus ofType A

The results were obtained using a specific antiserum of the PWDcircovirus produced during test 1 (cf. FIG. 1).

Type of Products Analyzed

The analyses were carried out on cell extracts of Sf9 cells obtainedafter infection by the recombinant baculovirus PCV ORF 1.

The culture of Sf9 cells was carried out in a 25 cm² Petri dishaccording to the standard culture methods for these cells. Aftercentrifugation, the cell pellets are taken up with 300 μl of PBS buffer(phosphate saline buffer).

Electrophoresis (PAGE-SDS)

The electrophoresis is carried out on the cell extracts of Sf9 cellsobtained previously on 5 samples (cf. Table 1 below) under the followingconditions:

-   % polyacrylamide gel: 8%; conditions: denaturing-   Voltage: 80 V; duration: 135 mn.

TABLE 1 Nature of the samples subjected to electrophoresis Well No. 1 23 4 5 Sample applied PM Raoul Raoul Raoul Raoul Rainbow 24 h 48 h 72 h96 h μl of sample 10 15 15 15 15 μl of Laemmli  0  5  5  5  5 4X Legendsto Table 1: Laemmli 4X: loading buffer PM Rainbow: molecular-weightmarkers (35, 52, 77, 107, 160 and 250 kD) Raoul 24 h, 48 h, 72 h and 96h: expression products of the ORF1 of the PWD circovirus of type A.

Western Blot

After electrophoresis, the bands obtained in the different wells aretransferred to nitrocellulose membrane for 1 h at 100 v in a TGM buffer(tris-glycine-methanol).

The Western blot is carried out under the following conditions:

-   -   1) Saturation with a solution containing 5% of skimmed milk;        0.05% of Tween 20 in a TBS 1×buffer (tris buffer saline) for 30        min.    -   2) 1st antibody:        -   10 ml of PWD anticircovirus antibody of type A are added            diluted to 1/100, then the reaction mixture is incubated for            one night at 4° C. Three washes of 10 min in TBS 1× are            carried out.    -   3) 2nd antibody:        -   10 ml of pig rabbit P164 antibody anti-immunoglobulins,            coupled to peroxidase (Dakopath), are added diluted to            1/100, then the reaction medium is incubated for 3 hours at            37° C. Three washes of 10 min in TBS 1× are carried out.    -   4) Visualization        -   The substrate 4-chloro-1-naphthol in the presence of            oxygenated water is used for visualization. Results

The results are shown in FIG. 7.

Kinetics of Appearance of Antibodies Specific for the REP RecombinantProtein of the PWD Circovirus of Type A Expressed in Baculovirus AfterInfection of Pigs by the PWD Circovirus of Type A (test 4, cf. FIG. 1)

After infection of the pigs, a sample of serum of each of the infectedpigs is taken at different periods expressed in the table by the date oftaking (carried out here in the same year) and is then analyzed byWestern blot.

The visualization of the specific antibodies is carried out in themanner described previously.

The results obtained are shown by Table 2 below.

TABLE 2 Kinetics of appearance of specific antibodies Sample Pigs 10/616/06 23/06 01/07 08/07 15/07 21/07 A3 1 Neg. Control 2 Neg. B2 Infec. 1Neg. Neg. Neg. + + ++ +++ RP+ 2 Neg. Neg. Neg. Neg. Neg. Neg. Neg. 3Neg. Neg. Neg. Neg. + + + 4 Neg. Neg. Neg. Neg. Neg. Neg. ++ Legends toTable 2: A3 control: uninfected control animals; B2 Infec. RP+: animalsinfected with pig kidney (PK) cells containing the circovirus; Neg.:negative; +, ++, +++: intensity scale of the positive reaction; 10/06,16/06, 23/06, 01/07, 08/07, 15/07, 21/07: dates expressed in day/monthon which the different withdrawals of serum were carried out.

Example 2 Cloning, Sequencing and Characterization of the Type B PWDCircovirus (PCVB)

The techniques used for cloning, sequencing and characterization of thetype B PWD circovirus (PCVB) are those used in Example 1 above for thetype A PWD circovirus (PCVA).

The nucleic acid sequence of the strand of (+) polarity of the genome ofthe PWD circovirus of type B (or PCVB) is represented by the sequenceSEQ ID No. 15 in the sequence listing, the nucleic acid sequence of thestrand of (−) polarity of the genome of the PWD circovirus of type B (orPCVB) being represented by the nucleic acid sequence 3′→5′ of FIG. 8 orby the sequence SEQ ID No. 19 (represented according to the orientation5′→3′) in the sequence listing.

The amino acid sequences, SEQ ID No. 24, SEQ ID No. 26 and SEQ ID No. 28of the sequence listing, respectively, represent the sequences of theproteins encoded by the nucleic sequences of the 3 open reading framesSEQ ID No. 23 (ORF′1), corresponding to the REP protein, SEQ ID No. 25(ORF′2) and SEQ ID No. 27 (ORF′3), determined from the sequence SEQ IDNo. 15 of the strand of (+) polarity or from the nucleic sequence SEQ IDNo. 19 of the strand of (−) polarity of the genome of the PWD circovirusof type B.

Example 3 Comparative Analysis of Nucleotide Sequences (ORF1, ORF2 andGenomic) and Amino Acid Sequences Encoded by the ORF1 and the ORF2 ofthe PWD Circoviruses of Type A (PCVA) and of Type B (PCVB)

The results expressed in % of homology are shown in Tables 3 and 4below.

TABLE 3 Compared analysis of the amino acid sequences % homology ORF1ORF2 PCVA/PCVB 80.4 56.2

TABLE 4 Compared analysis of the nucleotide sequences % homology GenomicORF1 ORF2 The remainder PCVA/PCVB 70.4 80.4 60.1 66.1

Example 4 Observation of the Disease and Reproduction of the DiseaseUnder Experimental Conditions

a) Test No. 1: Observation of the Disease

The objective is to take breeding animals at the start of disease and toplace them under experimental conditions to follow the progression ofthe pathology and describe all the clinical signs thereof. This firsttest was carried out on 3 breeding pigs aged 10 weeks of which 2 werealready ill (suffering from wasting), and on 3 other pigs aged 13 weeks,not having signs of disease. The clinical observation was spread over aperiod of 37 days. Two pigs of 10 weeks wasted rapidly (pigs 1 and 2,FIG. 9) and had to be painlessly killed 5 and 6 days after theirarrival. A single pig exhibited hyperthermia over 5 days and diarrhea.Two other pigs exhibited dyspnea and cough, of which one additionallyhad hyperthermia, greater than 41° C., for the two first days of itsstay. Another pig had retarded growth in the second week (pig 6, FIG.9), without any other clinical sign being recorded. On the lesionallevel, 5 pigs out of 6 exhibited macroscopic lesions of gray pneumonia,the sixth exhibited cicatricial lesions on the lung.

b) Test No. 2: Reproduction of the Disease from Inocula Prepared in FarmPigs.

The two sick pigs in test 1 served to prepare inocula which were testedin test 2 on specific-pathogen-free (SPF) pigs. The SPF pigs were aged 9weeks at the time of inoculation. The clinical and lesional results areshown in Table 5.

TABLE 5 Summary of the measurements carried out during experimentalreproduction of PWD. (The values of the control animals are reported inbrackets, the underlined values indicate a difference between infectedanimals and control animals) Test Measurement 2 3 4 5 6 7 Status of thepigs SPF SPF SPF SPF Conventional Conventional CNEVA field CNEVA CNEVAAge 9 weeks 6 weeks 5 weeks 5 weeks 5 weeks 6-7 weeks Number 4 6 12 8 88 Inoculation route Intratracheal route Intratracheal routeIntratracheal + Intratracheal + Intratracheal + Intratracheal +intramuscular route intramuscular route intramuscular routeintramuscular route Inoculum titer per pig ND* ND* 10^(4.53) TCID₅₀10^(4.53) TCID₅₀ 10^(4.53) TCID₅₀ 10^(4.53) TCID₅₀ per ml: 1 ml per ml:1 ml per ml: 1 ml per ml: 1 ml IM + 5 ml IT IM + 5 ml IT IM + 5 ml ITIM + 5 ml IT Start of hyperthermia 10 days 9-13 days 12-13 days 9-14days 8-12 days 12 days post-infection post-infection post-infectionpost-infection post-infection post-infection % of pigs in 100% 83% 92%100% 75% 88% hyperthermia** Number of days of 7 4.5 3.3 5.8 7.5 11.6hyperthermia per pig** Maximum 40.4 to 41.7° C. 40.6 to 42.3° C. 40.2 to41.6° C. 40.3 to 40.8° C. 40.6 to 42° C. 40.2 to 41.9° C.temperatures*** Hyperthermia**** % per week W1 3.5 (3.5) 17 (36) 7 (5)37 (17) 16 (17) 20 (28) W2  42 (3.5)  7 (13) 13 (1)  21 (3)  52 (10) 37(28) W3  35 (3.5) 33 (10) 28 (7)  62 (2)  34 (12) 79 (17) W4  21 (3.5)28 (7)  5 (0) 6 (3) 25 (22) 55 (3)  DMG: W1  928 (1053) 417 (357) 564(620) 650 (589) 401 (407) 509 (512) W2  678 (1028) 428 (617) 503 (718)612 (584) 294 (514) 410 (310) W3  661 (1000) 771 (642) 381 (657)520 (851) 375 (586) 435 (440) W4  786 (1100) 550 (657) 764 (778) 641(696) 473 (610) 451 (681) Contact pigs Yes to 100% Yes to 75% Not testedNot tested Not tested Not tested transmission % of pulmonary 25 75 0 2525 12 lesions % of ganglionic 17 33 67 25 50 12 lesions *ND: notdetermined, **hyperthermia when the temperature is greater than 40° C.,***range of maximum temperatures recorded at the individual level,****the percentage corresponds to the number of temperature recordingsgreater than 40° C. divided by the total number of temperaturerecordings in the week on all of the pigs.

In this test, there was no wasting, at the very most a retardation ofthe growth in the second, third or fourth week after infection. Thesedata illustrate that certain breeding conditions probably favor theexpression of the disease.

c) Tests No. 3 to No. 7: Reproduction of the Experimental Tests

The increase in the number of the experimental tests on pigs had themastering and better characterization of the experimental model as anobjective. All of the results are presented in Table 5.

Under the experimental conditions, PWD is thus characterized by a longincubation, of 8 to 14 days, true hyperthermia over 2 to 8 days, adecrease in food consumption and a retardation of the increase in weighton the second, third or fourth week post-infection. The lesional tableassociated with this clinical expression includes, in the main,ganglionic hypertrophy and lesions of pneumonia.

Conclusion

The perfection of this experimental model allows the direct etiologicalrole of the PWD circovirus in the disease to be indisputablydemonstrated. In addition, this model is an indispensable tool for theunderstanding of pathogenic mechanisms and the study of future vaccinecandidates.

Example 5 Demonstration of the Vaccine Composition Protective EfficacyProduced from Nucleic Fragments of PWD Circovirus Sequence

1) Animals Used for the Study

Piglets having the PWD disease, reproduced under experimental conditionsdescribed in paragraph c) of Example 4, were used in a protocol forevaluating the vaccine composition efficacy, comprising nucleicfragments of PWD circovirus sequence.

2) Tested Vaccine Composition and Vaccination Protocol

a) Components Used for the Study

The plasmids were obtained from the pcDNA3 plasmid of INVITROGENE

pcDNA3ORF-Plasmids

These plasmids are plasmids which do not carry a PWD circovirus nucleicacid insert and are used as a negative control plasmid.

pcDNA3ORF1+ Plasmid and pcDNA3ORF2+ Plasmid

The pcDNA3ORF1+ and pcDNA3ORF2+ plasmids are plasmids which carry anucleic acid insert of the sequence of the PWD circovirus of TYPE B, andan insert comprising the nucleic acid fragment SEQ ID No. 23 (ORF′1)coding for the Rep protein of sequence SEQ ID No. 24 and an insertcomprising the nucleic acid fragment SEQ ID No. 25 (ORF′2) coding forthe protein of sequence SEQ ID No. 26, probably corresponding to thecapsid protein, respectfully. These nucleic constructs further comprisethe ATG initiation codon of the coding sequence of the correspondingprotein.

GMCSF+ Plasmid

GM-CSF (granulocyte/macrophage colony stimulating factor) is a cytokinewhich occurs in the development, the maturation and the activation ofmacrophages, granulocytes and dendritic cells which present an antigen.The beneficial contribution of the GM-CSF in vaccination is consideredto be a cellular activation with, especially, the recruitment and thedifferentiation of cells which present an antigen.

This pcDNA3-GMCSF+ plasmid carries a nucleic acid insert coding for thegranulocyte/macrophage colony stimulation factor, the GM-CSF protein.

The gene coding for this GM-CSF protein was cloned and sequenced byInumaru et al. (Immunol. Cell Biol., 1995, 73 (5), 474-476). ThepcDNA3-GMCSF+ plasmid was obtained by Dr. B. Charley of NRA ofJouy-en-Josas (78, France).

Recombinant Baculoviruses

The so-called ORF-baculoviruses are viruses not carrying any insertcomprising a nucleic acid fragment capable of expressing a PWDcircovirus protein.

The so-called ORF1+ (BAC ORF1+) or ORF2+ (BAC ORF2+) baculoviruses arerecombinant baculoviruses carrying an insert comprising a nucleic acidfragment SEQ ID No. 23 (ORF′1) and an insert comprising the nucleic acidfragment SEQ ID No. 25 (ORF′2), respectively.

Adjuvant

The adjuvant supplied by the Seppic Company, a subsidiary of AIRLIQUIDE, is the adjuvant corresponding to the reference AIF SEPPIC.

b) Vaccination Protocol

Weaned piglets aged 3 weeks are divided into four batches A, B, C and Deach comprising 8 piglets.

Batches A, B and C, aged 3 weeks, each receive a first injection(injection M1) of 1 ml containing 200 micrograms of plasmids (naked DNA)in PBS, pH: 7.2, by the intramuscular route for each of the plasmidsmentioned below for each batch, then, at the age of 5 weeks, a secondinjection (injection M2) comprising these same plasmids. A thirdinjection is carried out simultaneously on the other side of the neck.This third injection comprises 1 ml of a suspension containing 5×10⁶cells infected by recombinant baculoviruses and 1 ml of AIF SEPPICadjuvant.

Batch A (F1) (Control Batch):

First Injection

pcDNA3ORF1-plasmid, pcDNA3ORF2-plasmid and GMCSF+ plasmid.

Second and Third Injection (Simultaneous)

pcDNA3ORF1-plasmid, pcDNA3ORF2-plasmid and GMCSF+ plasmid;

Cells transformed by baculoviruses not containing any nucleic acidinsert coding for a PWD circovirus protein;

AIF SEPPIC adjuvant.

Batch B (F2) (Control Batch):

First Injection

pcDNA3ORF1-plasmid, pcDNA3ORF2-plasmid and GMCSF+ plasmid;

Second and Third Injection (Simultaneous)

pcDNA3ORF1-plasmid, pcDNA3ORF2-plasmid and GMCSF+ plasmid;

Cells transformed by baculoviruses not containing any nucleic acidinsert coding for a PWD circovirus protein;

AIF SEPPIC adjuvant.

Batch C (F3):

First Injection

pcDNA3ORF1+ plasmid, pcDNA3ORF2+ plasmid and GMCSF+ plasmid;

Second and Third Injection (Simultaneous)

pcDNA3ORF1+ plasmid, pcDNA3ORF2+ plasmid and GMCSF+ plasmid;

Cells transformed by BAC ORF1+ and BAC ORF2+ recombinant baculovirusescapable of respectively expressing the Rep protein of sequence SEQ IDNo. 24 and the protein of sequence SEQ ID No. 26 of the PWD circovirusof TYPE B. Batch D (F4) (control batch): no injection

The batches of piglets B, C and D are infected (tested) at the age of 6weeks although batch A is not subjected to the test.

3) Observation of the Batches

-   counting of coughing/sneezing: 15 minutes/batch/day;-   consistency of fecal matter: every day;-   regular recordings: weekly taking of blood, weighing;-   weighing of food refuse: 3 times per week;-   calculation of the daily mean gain in weight (dmg);

The daily mean gains were calculated for each of the batches over aperiod of 28 days following testing (cf. FIG. 10), an intermediatecalculation of the dmg was likewise carried out for each of the batchesover the first and second periods of 14 days. The results obtained arereported below in Table 6.

TABLE 6 Daily mean gains F1 F2 F3 F4 d0-d14 411 g 450 g 511 g 461 gd14-d28  623 g 362 g 601 g 443 g d0-d28 554 g 406 g 556 g 452 g

-   Measurement of hyperthermia

The measurement of hyperthermia, of greater than 41° C. (cf. FIG. 11)and greater than 40.2° C., was carried out for each of the batches overa total period of 28 days following testing. The results obtained,corresponding to the ratio expressed as a percentage between the numberof temperature recordings of greater than 41° C. (or greater than 40.2°C.) and the total number of temperature recordings carried out on all ofthe pigs per one-week period are reported below in Tables 7 and 8,respectively, for the hyperthermia measurements of greater than 41° C.and greater than 40.2° C.

TABLE 7 Hyperthermia > 41° C. F1 F2 F3 F4 W1 4.1 0 0 0 W2 10.7 16. 0 8.9W3 4.7 27. 0 45. W4 0 0 0 7.5

TABLE 8 Hyperthermia > 40.2 F1 F2 F3 F4 W1 29.1 10.41 29.1 20.8 W2 28.539.2 10.7 37.5 W3 14.3 68.7 25.0 81.2 W4 3.3 17.5 20.0 55

4) Conclusion

The recordings carried out clearly show that the animals which receivedthe three injections of a vaccine composition comprising nucleic acidfragments of PWD circovirus according to the invention and/or capable ofexpressing recombinant proteins of PWD circovirus, in particular of typeB, did not exhibit hyperthermia (cf. FIG. 10). These animalsadditionally did not experience a decline in their growth, the dmgsbeing comparable to those of uninfected control animals (cf. FIG. 9).They did not exhibit any particular clinical sign.

These results demonstrate the efficacious protection of the pigletsagainst infection with a PWD circovirus of the invention, the primaryagent responsible for PWD or FPW, provided by a vaccine compositionprepared from a nucleic acid fragment of the nucleic sequence of PWDcircovirus according to the invention, in particular of type B, and/orfrom recombinant proteins encoded by these nucleic acid fragments.

These results in particular show that the proteins encoded by the ORF1and ORF2 of PWD circovirus according to the invention are immunogenicproteins inducing an efficacious protective response for the preventionof infection by a PWD circovirus.

Example 6 Serological Diagnosis of PWD Circovirus by ImmunodeterminationUsing Recombinant Proteins or Synthetic Peptides of PWD Circovirus

A. Serological Diagnosis with Recombinant Proteins

The identification and the sequencing of porcine PWD circovirus allowrecombinant proteins of PWD circovirus to be produced by the techniquesof genetic recombination well known to the person skilled in the art.Using these techniques, recombinant proteins encoded, in particular, bythe ORF′2 of the PWD circovirus, type B, were expressed by transformedSf9 insect cells and then isolated.

These recombinant proteins encoded by the ORF′2 are extracted, afterculture of the transformed Sf9 cells, by thermal cell lysis by means of3 cycles of freezing/thawing to −70° C./+37° C. Healthy Sf9 cells ornontransformed control Sf9 cells are also lysed.

Two antigenic fractions originating from nontransformed control Sf9cells and Sf9 cells expressing the ORF′2 are precipitated at 4° C. by a60% plus or minus 5% saturated ammonium sulfate solution. Determinationof total proteins is carried out with the aid of the Biorad kit. 500 ngof control Sf9 proteins and of semipurified Sf9 proteins expressing theORF′2, in solution in 0.05 M bicarbonate buffer pH 9.6, are passivelyadsorbed at the bottom of 3 different wells of a Nunc Maxisorpmicroplate by incubation for one night at +4° C.

The reactivity of pig sera with respect to each of these antigenicfractions is evaluated by an indirect ELISA reaction of which theexperimental protocol is detailed below:

-   Saturation step: 200 μl/well of PBS1×/3% semi-skimmed milk, 1 h 30    incubation at 37° C.-   Washing: 200 μl/well of PBS1×/Tween 20: 0.05%, 3 rapid washes.-   Serum incubation step: 100 μl/well of serum diluted to 1/100 in    PBS1×/semi-skimmed milk, 1%/Tween 20: 0.05%, 1 h incubation at 37°    C.-   Washing: 200 μl/well of PBS1×/Tween 20: 0.05%, 2 rapid washes    followed by 2 washes of 5 min.-   Conjugate incubation step: 50 μl/well of rabbit anti-pig conjugate    diluted to 1/1000 in PBS1×/semi-skimmed milk, 1%/Tween 20: 0.05%, 1    h incubation at 37° C.-   Washing: 200 μl/well of PBS1×/Tween 20: 0.05%, 2 rapid washes    followed by 2 washes of 5 min.-   Visualization step: 100 μl/well of OPD substrate/citrate    buffer/H₂O₂, 15 min incubation at 37° C.-   Termination: 50 μl/well of 1 N H₂SO₄.-   Read optical density in a spectrophotometer at 490 nm.

Results

The results obtained are shown below in Table 9.

TABLE 9 Reactivity of Pig Serum Reactivity of Pig Serum not inoculatedwith inoculated with Antigens Circovirus Circovirus Purified Sf9 control0.076 0.088 Sf9 expressing purified 0.071 1.035 ORF′2

The results are expressed in optical density measured in aspectrophotometer at 490 nm during analysis by ELISA of the reactivityof pig sera which are or are not inoculated with the type B PWDcircovirus according to the protocol indicated above.

B. Serological Diagnosis by Synthetic Peptide

The epitopic mapping of the proteins encoded, for example, by thenucleic sequences ORF1 and ORF2 of the two types of PWD circovirus(types A and B) additionally allowed immunogenic circoviral epitopes tobe identified on the proteins encoded by the nucleic sequences ORF′1 andORF′2 as well as the specific epitopes of the protein encoded by thenucleic acid sequence ORF′2 of the type B PWD circovirus. Four specificepitopes of the type B PWD circovirus and one epitope common to the twotypes of PWD circovirus situated on the protein encoded by the nucleicsequence ORF′2 were synthesized in peptide form. The equivalent peptidesin the circovirus of type A were likewise synthesized. All peptides wereevaluated as diagnostic antigens within the context of carrying out aserological test.

Results

The results obtained are shown in Table 10, below.

TABLE 10 Results of the evaluation as a diagnostic antigen of syntheticpeptides encoded by the nucleic sequences ORF2 and ORF′2 of PWDcircovirus of type A and B. Infected pig serum Type reactivityCircovirus B PWD SPF Conventional Conventional Epitopic Peptidecircovirus Position AA sequence D0/D54 1 D0/D42 2 D0/D42 specificity SEQID NO: 29 121 B 71-85 VDMMRFNINDFLPPG +/−, +++ +/−, +++ −, +++Circovirus SEQ ID NO: 55 177 B 70-84 NVNELRFNIGQFLPP +/−, + +/−, +/−+/−, − B SEQ ID NO: 30 132 B 115-129 QGDRGVGSSAVILDD +/−, +/− ++, +++/−, + Circovirus SEQ ID NO: 56 188 A 114-127 TSNQRGVGSTVVIL +/−, − −,+/− +/−, +/− B SEQ ID NO: 31 133 B 119-134 GVGSSAVILDDNVFTK −, ++ ++,+++ +/−, ++ SEQ ID NO: 57 189 A 118-132 RGVGSTVVILDANFV +/−, − −, +/−+/−, +/− SEQ ID NO: 58 146 B 171-185 FTIDYFQPNNKRNQL −, +/− −, ++ −, ++Circovirus SEQ ID NO: 59 202 A 170-184 DQTIDWFQPNNKRNQ +++, +++ +/−, +++, ++ A&B SEQ ID NO: 32 152 B 195-209 VDHVGLGTAFENSIY −, ++ +++, ++++/−, + Circovirus SEQ ID NO: 60 208 A 194-208 NVEHTGLGYALQNAT −, − −, −−, − B +/−, +, ++, +++. Increasing intensities of the reactivitiesobserved in Spot peptides on a nitrocellulose membrane. The porcine seratested are from animals experimentally infected with the circovirus oftype B within the animal houses of the CNEVA. Samples are taken from theanimals before inoculation on d0 and 42 days or 54 days afterinoculation, on d42, d54.

Example 7 Characterization of the Specific Epitopes of the PWDCircovirus of type B

The proteins encoded by the ORF2 of the porcine circoviruses of type Aand B were chosen for this study. For each of the ORF2s (types A and B),56 peptides of 15 amino acids which overlap every 4 amino acids weresynthesized, thus covering the whole of the protein (cf. Table 11below).

TABLE 11 Sequence of amino acids of the 56 peptides of 15 amino acidssynthesized from the nucleic sequence ORF′2 (type B) and ORF2 (type A)of PWD circovirus with their corresponding spot number (cf. FIG. 12)Type B ORF′2 Type A ORF2 Spot No. Sequence Spot No. Sequence SEQ ID NO:171 104 MTYPRRRYRRRRHRP SEQ ID NO: 175 160 MTWPRRRYRRRRTRP SEQ ID NO:172 105 RRRYRRRRHRPRSHL SEQ ID NO: 176 161 RRRYRRRRTRPRSHL SEQ ID NO:173 106 RRRRHRPRSHLGQIL SEQ ID NO: 177 162 RRRRTRPRSHLGNIL SEQ ID NO: 61107 HRPRSHLGQILRRRP SEQ ID NO: 84 163 TRPRSHLGNILRRRP SEQ ID NO: 62 108SHLGQILRRRPWLVH SEQ ID NO: 85 164 SHLGNILRRRPYLVH SEQ ID NO: 63 109QILRRRPWLVHPRHR SEQ ID NO: 86 165 NILRRRPYLVHPAFR SEQ ID NO: 64 110RRPWLVHPRHRYRWR SEQ ID NO: 87 166 RRPYLVHPAFRNRYR SEQ ID NO: 65 111LVHPRHRYRWRRKNG SEQ ID NO: 88 167 LVHPAFRNRYRWRRK SEQ ID NO: 66 112RHRYRWRRKNGIFNT SEQ ID NO: 89 168 AFRNRYRWRRKTGIF SEQ ID NO: 67 113RWRRKNGIFNTRLSR SEQ ID NO: 90 169 RYRWRRKTGIFNSRL SEQ ID NO: 68 114KNGIFNTRLSRTFGY SEQ ID NO: 91 170 RRKTGIFNSRLSREF SEQ ID NO: 69 115FNTRLSRTFGYTVKR SEQ ID NO: 92 171 GIFNSRLSREFVLTI SEQ ID NO: 70 116LSRTFGYTVKRTTVR SEQ ID NO: 93 172 SRLSREFVLTIRGGH SEQ ID NO: 71 117FGYTVKRTTVRTPSW SEQ ID NO: 94 173 REFVLTIRGGHSQPS SEQ ID NO: 72 118VKRTTVRTPSWAVDM SEQ ID NO: 95 174 LTIRGGHSOPSWNVN SEQ ID NO: 73 119TVRTPSWAVDMMRFN SEQ ID NO: 96 175 GGHSQPSWNVNELRF SEQ ID NO: 74 120PSWAVDMMRFNINDF SEQ ID NO: 97 176 QPSWNVNELRFNIGO SEQ ID NO: 29 121VDMMRFNINDFLPPG SEQ ID NO: 98 177 NVNELRFNIGQFLPP SEQ ID NO: 75 122RFNINDFLPPGGGSN SEQ ID NO: 99 178 LRFNIGQFLPPSGGT SEQ ID NO: 76 123NDFLPPGGGSNPRSV SEQ ID NO: 100 179 IGQFLPPSGGTNPLP SEQ ID NO: 77 124PPGGGSNPRSVPFEY SEQ ID NO: 101 180 LPPSGGTNPLPLPFQ SEQ ID NO: 78 125GSNPRSVPFEYYRIR SEQ ID NO: 102 181 GGTNPLPLPFQYYRI SEQ ID NO: 79 126RSVPFEYYRIRKVKV SEQ ID NO: 103 182 PLPLPFQYYRIRKAK SEQ ID NO: 80 127FEYYRIRKVKVEFWP SEQ ID NO: 104 183 PFQYYRIRKAKYEFY SEQ ID NO: 81 128RIRKVKVEFWPCSPI SEQ ID NO: 105 184 YRIRKAKYEFYPRDP SEQ ID NO: 82 129VKVEFWPCSPITQGD SEQ ID NO: 106 185 KAKYEFYPRDPITSN SEQ ID NO: 83 130FWPCSPITQGDRGVG SEQ ID NO: 107 186 EFYPRDPITSNQRGV SEQ ID NO: 174 131SPITQGDRGVGSSAV SEQ ID NO: 108 187 RDPITSNQRGVGSTV SEQ ID NO: 30 132QGDRGVGSSAVILDD SEQ ID NO: 109 188 TSNQRGVGSTVVILD SEQ ID NO: 31 133GVGSSAVILDDNFVT SEQ ID NO: 136 189 RGVGSTVVILDANFV SEQ ID NO: 111 134SAVILDDNFVTKATA SEQ ID NO: 137 190 STVVILDANFVTPST SEQ ID NO: 112 135LDDNFVTKATALTYD SEQ ID NO: 138 191 ILDANFVTPSTNLAY SEQ ID NO: 113 136FVTKATALTYDPYVN SEQ ID NO: 139 192 NFVTPSTNLAYDPYI SEQ ID NO: 114 137ATALTYDPYVNYSSR SEQ ID NO: 140 193 PSTNLAYDPYINYSS SEQ ID NO: 115 138TYDPYVNYSSRIITIT SEQ ID NO: 141 194 LAYDPYINYSSRHTI SEQ ID NO: 116 139YVNYSSRHTITQPFS SEQ ID NO: 142 195 PYINYSSRHTIRQPF SEQ ID NO: 117 140SSRHTITQPFSYHSR SEQ ID NO: 143 196 YSSRIITIRQPFTYHS SEQ ID NO: 118 141TITQPFSYHSRYFTP SEQ ID NO: 144 197 HTIRQPFTYHSRYFT SEQ ID NO: 119 142PFSYHSRYFTPKPVL SEQ ID NO: 145 198 QPFTYHSRYFTPKPE SEQ ID NO: 120 143HSRYFTPKPVLDFTI SEQ ID NO: 146 199 YHSRYFTPKPELDQT SEQ ID NO: 121 144FTPKPVLDFTIDYYFQ SEQ ID NO: 147 200 YFTPKPELDQTIDWF SEQ ID NO: 122 145PVLDFTIDYFQPNNK SEQ ID NO: 148 201 KPELDQTIDWFQPNN SEQ ID NO: 123 146FTIDYFQPNNKRNQL SEQ ID NO: 149 202 DQTIDWFQPNNKRNQ SEQ ID NO: 124 147YFQPNNKRNQLWLRL SEQ ID NO: 150 203 DWFQPNNKRNQLWLH SEQ ID NO: 125 148NNKRNQLWLRLQTAG SEQ ID NO: 151 204 PNNKRNQLWLHLNTH SEQ ID NO: 126 149NQLWLRLQTAGNVDH SEQ ID NO: 152 205 RNQLWLHLNTHTNVE SEQ ID NO: 127 150LRLQTAGNVDHVGLG SEQ ID NO: 153 206 WLHLNTHTNVEHTGL SEQ ID NO: 128 151TAGNVDHVGLGTAFE SEQ ID NO: 154 207 NTHTNVEHTGLGYAL SEQ ID NO: 32 152VDHVGLGTAFENSIY SEQ ID NO: 155 208 NVEHTGLGYALQNAT SEQ ID NO: 129 153GLGTAFENSIYDQEY SEQ ID NO: 156 209 TGLGYALQNATTAQN SEQ ID NO: 130 154AFENSIYDQEYNIRV SEQ ID NO: 157 210 YALQNATTAQNYVVR SEQ ID NO: 131 155SIYDQEYNIRVTMYV SEQ ID NO: 158 211 NATTAQNYVVRLTIY SEQ ID NO: 132 156QEYNIRVTMYVQFRE SEQ ID NO: 159 212 AQNYVVRLTIYVQFR SEQ ID NO: 133 157IRVTMYVQFREFNFK SEQ ID NO: 160 213 VVRLTIYVQFREFIL SEQ ID NO: 134 158MYVQFREFNFKDPPL SEQ ID NO: 161 214 TIYVQFREFILKDPL SEQ ID NO: 135 159VQFREFNFKDPPLNP SEQ ID NO: 162 215 YVQFREFILKDPLNE

These peptides were synthesized according to the “spot” method whichconsists of simultaneous synthesis of a large number of peptides on acellulose solid support, each site of synthesis of a peptideconstituting a spot (Synt:em, NIMES). This method involves orientationof the peptides on the plate, these being fixed covalently by thecarboxy-terminal end. A spot represents approximately 50 nmol ofpeptide.

The reference of the spots and corresponding peptide sequences is givenin Table 11.

These membranes were used for immunoreactivity tests with respect toserum of SPF pigs which were or were not infected experimentally withthe type B PWD circoviral strain as well as with respect to sera ofinfected pigs from conventional farms (conventional farms 1 or 2). Thisstudy allowed specific immunoreactive peptides of the circovirus of typeB corresponding to the spots No. 121, No. 132, No. 133 and No. 152(respectively of amino acid sequences SEQ ID No. 29, SEQ ID No. 30, SEQID No. 31 and SEQ ID No. 32) to be demonstrated. An illustration isshown in FIG. 12 where the membranes are visualized with an infected pigserum coming from a conventional farm. Nonspecific immunoreactivepeptides of type [lacuna] were likewise demonstrated, among which weshall keep the peptide No. 146 SEQ ID No. 123 which is stronglyimmunogenic.

A comparison between the peptide sequences of circoviruses of type A andB (FIG. 13) indicates a divergence ranging from 20 to 60% for thespecific immunoreactive peptides of the type B, and a weaker divergence(13%) between the nonspecific peptides.

Example 8 Protection of Swine From Post-Weaning Multisystemic WastingSyndrome (PMWS) Conferred by Procine Circovirus Type B (PCV-B) ORF′2Protein.

The ORF′1-encoded protein (REP) and ORF′2-encoded putative capsidprotein of PCV-B were expressed, either in insect cells by recombinantbaculovirus vectors, or in mammalian cell lines by transfection withplasmidic expression vectors. These two circovirus-derived proteins weredetectable in both expression systems. As evaluated by weight gains,hyperthermia and absence of lesions following challenge, the pigs wereprotected against a virulent circovirus challenge after one first DNAimmunization with plasmids directing ORF′2 protein and GM-CSF expressionand a second injection, 15 days later, with the same plasmid preparationplus the ORF′2 recombinant protein. A lower level of protection wasobserved when the pigs were vaccinated with ORF′1 protein, as opposed topigs vaccinated with ORF′2 protein.

A. Development of an Experimental Model of PMWS in Swine:

Eight 3 week-old SPF pigs were inoculated intratracheally (5 ml) andintramuscularly (1 ml).

B. Production and Control of PCV-B Plasmids:

PCV-B ORF′1 and ORF′2 genes, isolated from PCV-B challenge strain, wascloned into vector plasmid pcDNA3.1. All constructs were validatedthrough a partial sequencing of the PCV-B genes in the final plasmidsand expression control by immunoperoxidase on PK15 cells respectivelytransfected with each plasmid, using swine polyclonal antibodies.

Plasmid encoding GM-CSF has been co-administered.

C. Construction of Recombinant Baculoviruses:

ORF′1 and ORF′2 proteins were expressed under polyhedrin promotercontrol. Recombinant proteins were detected by western-blot using swinepolyclonal antibodies.

D. Vaccination and Challenge:

Four groups of 7 pigs were vaccinated intramuscularly at day 0 (Do), twoweeks later, they received the same plasmid preparation plus therecombinant baculovirus.

E. Monitoring:

All groups of pigs were housed in isolated experimental units with airfiltration and low air pressure. Clinical observations and rectaltemperatures were recorded every day. The pigs were weighed weekly.

F. Conclusions

Expression of PCV-B ORF′2 or PCV-B ORF′1 in swine resulted in asignificantly enhanced level of protection as evaluated by weightevolution and body temperature evolution following challenge with PCV-Bcircovirus. These results are summarized in FIGS. 14 and 15.

The invention described herein may be embodied in other specific formswithout departing from the spirit or essential characteristics thereof.The specific embodiments previously described are therefore to beconsidered as illustrative of, and not limiting, the scope of theinvention. Additionally, the disclosure of all publications and patentapplications cited above and below, including International PatentApplication No. PCT/FR98/02634, filed Dec. 4, 1998, and published asInternational Publication No. WO 99/29871 on Jun. 17, 1999, areexpressly incorporated herein by reference in their entireties to thesame extent as if each were incorporated by reference individually.

BIBLIOGRAPHIC REFERENCES

-   Allan, G. M. et al., 1995, Vet. Microbiol., 44: 49-64.-   Barany, F., 1911, PNAS. USA, 88: 189-193.-   Boulton, L. H. et al., 1997, J. Gen. Virol., 78 (Pt 6), 1265-1270.-   Buckholz, R. G., 1993, Yeast systems for the expression of    heterologous gene products. Curr. Op. Biotechnology 4: 538-542.-   Burg, J. L. et al., 1996, Mol. and Cell. Probes, 10: 257-271.-   Chu, B. C. F. et al., 1986, NAR, 14: 5591-5603.-   Chu, P. W. G. et al., 1993, Virus Research, 27: 161-171.-   Clark, E. G., 1997, American Association of Swine Practitioners,    499-501.-   Daft, B. et al., 1996, American Association of Veterinary Laboratory    Diagnosticians, 32.-   Derse, D. et al., 1995, J. Virol., 69(3): 1907-1912.-   Duck, P. et al., 1990, Biotechniques, 9: 142-147.-   Dulac, G. C. et al., 1989, Can. J. Vet. Res., 53: 431-433.-   Edwards, C. P., and Aruffo, A., 1993, Current applications of COS    cell based transient expression systems. Curr. Op. Biotechnology 4:    558-563.-   Edwards, S. et al., 1994, Vet. Rec., 134: 680-681.-   Erlich, H. A., 1989, In PCR Technology. Principles and Applications    for DNA Amplification. New York: Stockton Press.-   Felgner, et al., 1987, Proc. Natl. Acad. Sci., 84: 7413.-   Fontes, E. P. B. et al., 1994, J. Biol. Chem., Vol. 269, No. 11:    8459-8465.-   Fraley et al., 1980, J. Biol. Chem., 255: 10431.-   Guateli, J. C. et al., 1990, PNAS. USA, 87: 1874-1878.-   Hackland, A. F. et al., 1994, Arch. Virol., 139: 1-22.-   Hanson, S. F. et al., 1995, Virology, 211: 1-9.-   Harding, J. C., 1997, American Association of Swine Practitioners,    503.-   Harding, R. M. et al., 1993, Journal of General Virology, 74:    323-328.-   Harding, J. C. and Clark, E. G., 1997, Swine Health and Production,    Vol. 5, No. 5: 201-203.-   Heyraud-Nitschke, F. et al., 1995, Nucleic Acids Research, Vol. 23,    No. 6.-   Horner, G. W., 1991, Surveillance 18(5): 23.-   Houben-Weyl, 1974, in Methode der Organischen Chemie, E. Wunsch Ed.,    Volume 15-I and 15-II, Thieme, Stuttgart.-   Huygen, K. et al., 1996, Nature Medicine, 2(8): 893-898.-   Innis, M. A. et al., 1990, in PCR Protocols. A guide to Methods and    Applications, San Diego, Academic Press.-   Kaneda, et al., 1989, Science, 243: 375.-   Kievitis, T. et al., 1991, J. Virol. Methods, 35: 273-286.-   Kohler, G. et al., 1975, Nature, 256(5517): 495-497.-   Kwoh, D. Y. et al., 1989, PNAS. USA, 86: 1173-1177.-   Ladany, S. et al., 1989, J. Clin. Microbiol. 27: 2778-2783.-   Lazarowitz, S. G. et al., 1989, The EMBO Journal, Vol. 8 No. 4:    1023-1032.-   Luckow, V. A., 1993, Baculovirus systems for the expression of human    gene products. Curr. Op. Biotechnology 4: 564-572.-   Mankertz, A. et al., 1997, J. Virol., 71: 2562-2566.-   Matthews, J. A. et al., 1988, Anal. Biochem., 169: 1-25.-   McNeilly, F. et al., 1996, Vet. Immunol. Immunopathol., 49: 295-306.-   Meehan, B. M. et al., 1997, J. Gen. Virol. 78: 221-227.-   Merrifield, R. D., 1966, J. Am. Chem. Soc., 88(21): 5051-5052.-   Midoux, 1993, Nucleic Acids Research, 21: 871-878.-   Miele, E. A. et al., 1983, J. Mol. Biol., 171: 281-295.-   Murphy, F. A. et al., 1995, Sixth Report of the International    Committee on Taxonomy of Viruses. Springer-Verlag Wien New York.-   Nayar, G. P. et al., 1997, Can. Vet. J. 38(6): 385-386.-   Olins, P. O., and Lee, S. C., 1993, Recent advances in heterologous    gene expression in E. coli. Curr. Op. Biotechnology 4: 520-525.-   Pagano et al., 1967, J. Virol., 1: 891.-   Rolfs, A. et al., 1991, In PCR Topics. Usage of Polymerase Chain    reaction in Genetic and Infectious Disease. Berlin: Springer-Verlag.-   Sambrook, J. et al., 1989, In Molecular cloning: A Laboratory    Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory    Press.-   Sanchez-Pescador, R., 1988, J. Clin. Microbiol., 26(10): 1934-1938.-   Segev D., 1992, in “Non-radioactive Labeling and Detection of    Biomolecules”. Kessler C. Springer Verlag, Berlin, N.Y.: 197-205.-   Shiver, J. W., 1995, in Vaccines 1995, eds Chanock, R.M. Brown, F.    Ginsberg, H.S. & Norrby, E., pp. 95-98, Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, N.Y.-   Tascon, R. E. et al., 1996, Nature Medicine, 2(8): 888-892.-   Tischer, I. et al., 1982, Nature, 295: 64-66.-   Tischer, I. et al., 1986, Arch. Virol., 91: 271-276.-   Tischer, I. et al., 1988, Zentralbl Bakteriol Mikrobiol Hyg [A] 270:    280-287.-   Tischer, I. et al., 1995, Arch. Virol., 140: 737-743.-   Urdea, M. S., 1988, Nucleic Acids Research, II: 4937-4957.-   Walker, G. T. et al., 1992, NAR 20: 1691-1696.-   Walker, G. T. et al., 1992, PNAS. USA, 89: 392-396.-   White, B. A. et al., 1997, Methods in Molecular Biology, 67, Humana    Press, Towota.-   Zhao, T. M. et al., 1996, Proc. Natl. Acad. Sci., USA 93(13):    6653-6648.

1. A vaccine comprising a nucleotide sequence of the genome of Porcine circovirus type B, or a homologue or fragment thereof, and an acceptable pharmaceutical or veterinary vehicle, wherein the nucleotide sequence is selected from SEQ ID No. 15 or SEQ ID No.
 19. 2. The vaccine of claim 1, wherein the homologue has at least 80% sequence identity to SEQ ID No. 15 or SEQ ID No.
 19. 3. A vaccine comprising a polypeptide encoded by a nucleotide sequence of the genome of Porcine circovirus type B, or a homologue or fragment thereof, and an acceptable pharmaceutical or veterinary vehicle.
 4. The vaccine of claim 3, wherein the homologue has at least 80% sequence identity to SEQ ID No. 15 or SEQ ID No.
 19. 5. The vaccine of claim 3, wherein the nucleotide sequence is selected from SEQ ID No. 23 or SEQ ID No. 25, or a homologue or fragment thereof.
 6. The vaccine of claim 5, wherein the homologue has at least 80% sequence identity to SEQ ID No. 23 or SEQ ID No.
 25. 7. The vaccine of claim 5, wherein the nucleotide sequence is SEQ ID No.
 25. 8. The vaccine of claim 3, wherein the polypeptide has the amino acid sequence of SEQ ID No. 24 or SEQ ID No.
 26. 9. The vaccine of claim 8, wherein the polypeptide has the amino acid sequence of SEQ ID No.
 26. 10. The vaccine of claim 3, wherein the homologue has at least 80% sequence identity to SEQ ID No. 24 or SEQ ID No.
 26. 11. The vaccine of claim 10, wherein the homologue has at least 80% sequence identity to SEQ ID No.
 26. 12. The vaccine of claim 3, wherein the polypeptide has the amino acid sequence of SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, or SEQ ID No.
 32. 13. A vaccine comprising a vector and an acceptable pharmaceutical or veterinary vehicle, the vector comprising a nucleotide sequence of the genome of Porcine circovirus type B, or a homologue or fragment thereof.
 14. A vaccine according to claim 13, further comprising a gene coding for an expression product capable of inhibiting or retarding the establishment or development of a genetic or acquired disease.
 15. A vaccine comprising a cell and an acceptable pharmaceutical or veterinary vehicle, wherein the cell is transformed with a nucleotide sequence of the genome of Porcine circovirus type B, or a homologue or fragment thereof.
 16. A vaccine comprising a pharmaceutically acceptable vehicle and a single polypetide, wherein the single polypeptide consists of SEQ ID No.
 26. 